Peptides and peptidomimetics in combination uses and treatments for cancer patient subpopulations

ABSTRACT

This invention provides compounds including peptides and peptidomimetics that can be used to treat cell proliferative disorders, such as those associated with benign and malignant tumor cells. While the invention is not limited to any particular mechanism, the compounds of the invention appear to function at least in part by inhibiting G2 cell cycle checkpoint. Thus, invention compounds can be used to inhibit cell growth alone or be used in combination with a nucleic acid damaging treatment to inhibit cell growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/838,777 filed Jun. 24, 2013, which application is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Nov. 11, 2014, isnamed SubSeqCanbas0432881_ST25.txt and is 44,672 bytes in size.

TECHNICAL FIELD

This invention relates to compounds including peptides andpeptidomimetics having anti-cell proliferative activity alone, and incombination with treatments that either directly or indirectly damagenucleic acid (e.g., DNA). The invention compounds are therefore usefulfor inhibiting cell proliferation and, as such, for treating cellproliferative disorders including cancer.

INTRODUCTION

The cell cycle comprises S phase (DNA replication), M phase (mitosis),and two gap phases (G1 and G2 phases) between S and M phases.Checkpoints in the cell cycle ensure accurate progression, such asmonitoring the state of DNA integrity, DNA replication, cell size, andthe surrounding environment (Mailer, J. L. Curr. Opin. Cell Biol., 3:26(1991)). It is especially important for multi-cellular organisms tomaintain integrity of genome, and there are multiple checkpoints thatmonitor the state of genome. Among them are G1 and G2 checkpointsexisting before DNA replication and mitosis, respectively. It is crucialto correct DNA damage before entering S phase, because once damaged DNAis replicated it often gives rise to mutations (Hartwell, L. Cell,71:543 (1992)). Progression through G1 and G2 checkpoints withoutrepairing extensive DNA damage induces apoptosis and/or catastrophe.

Most cancer cells carry abnormalities in G1 checkpoint-related proteinssuch as p53, Rb, MDM-2, p16^(INK4) and p19^(ARF) (Levine, A. J. Cell,88:323 (1997)). Alternatively, mutations can cause over-expressionand/or over activation of oncogene products, e.g., Ras, MDM-2 and cyclinD, which reduce the stringency of G1 checkpoint. In addition to thesemutations, excessive growth factor signaling can be caused by the overexpression of growth factors and can reduce the stringency of G1checkpoint. Together with loss and gain-of-function mutations,continuous activation of growth factor receptors or downstreamsignal-transducing molecules can cause cell transformation by overridingthe G1 checkpoint. Abrogated G1 checkpoint contributes to highermutation rates and the many mutations observed in cancer cells. As aresult, most cancer cells depend on G2 checkpoint for survival againstexcessive DNA damage (O'Connor and Fan, Prog. Cell Cycle Res., 2:165(1996)).

The mechanism that promotes the cell cycle G2 arrest after DNA damage isbelieved to be conserved among species from yeast to human. In thepresence of damaged DNA, Cdc2/Cyclin B kinase is kept inactive becauseof inhibitory phosphorylation of threonine-14 and tyrosine-15 residueson Cdc2 kinase or the protein level of Cyclin B is reduced. At the onsetof mitosis, the dual phosphatase Cdc25 removes these inhibitoryphosphates and thereby activates Cdc2/Cyclin B kinase. The activation ofCdc2/Cyclin B is equivalent to the onset of M phase.

In fission yeast, the protein kinase Chk1 is required for the cell cyclearrest in response to damaged DNA. Chk1 kinase acts downstream ofseveral rad gene products and is modified by the phosphorylation uponDNA damage. The kinases Rad53 of budding yeast and Cds1 of fission yeastare known to conduct signals from unreplicated DNA. It appears thatthere is some redundancy between Chk1 and Cds1 because elimination ofboth Chk1 and Cds1 culminated in disruption of the G2 arrest induced bydamaged DNA. Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 andpromote Rad24 binding to Cdc25, which sequesters Cdc25 to cytosol andprevents Cdc2/Cyclin B activation. Therefore Cdc25 appears to be acommon target of these kinases implying that this molecule is anindispensable factor in the G2 checkpoint.

In humans, both hChk1, a human homologue of fission yeast Chk1, andChk2/HuCds1, a human homologue of the budding yeast Rad53 and fissionyeast Cds1, phosphorylate Cdc25C at serine-216, a critical regulatorysite, in response to DNA damage. This phosphorylation creates a bindingsite for small acidic proteins 14-3-3s, human homologues of Rad24 andRad25 of fission yeast. The regulatory role of this phosphorylation wasclearly indicated by the fact that substitution of serine-216 to alanineon Cdc25C disrupted cell cycle G2 arrest in human cells. However, themechanism of G2 checkpoint is not fully understood.

Tumor microenvironment also plays a role in the prevention or promotionof cancer cell growth, invasion, metastasis and anti-tumor immunity,which affects patient prognosis. Macrophages, once expected to workagainst cancer cells, have been indicated to play both inhibitory andpromoting roles in the tumor development. Macrophages with classicalanti-tumor phenotype, referred to as M1, are pro-inflammatory, and thosewith pro-tumor and anti-inflammatory types are referred to as M2 with atleast three major subtypes within this category (Martinez and Gordon,F1000Prime Reports 6:13 (2014)).

Neutrophil extracellular traps (NETs) represent another element of thetumor microenvironment along with leukocytes. While the formation ofNETs are useful for neutrophils to fight against invading microorganismsthey may contribute to deep vein thrombosis (DVT) (Martinnod and Wanger,Blood (2013)) and tumor cell metastasis (Cools-Lartigue, J., et al. J.Clin. Invest. (2013)) in cancer patients. Thus, NETs may adverselyaffect patient survival. DVT is common and potentially lethal in cancerpatients, and leukocytosis (which leads to high WBC) is a major riskfactor (Pabinger, I., et al. Blood 122:12 (2013); Blix, K., et al. PLOSOne 4:8 (2013); Wang, T. F., et al. Thromb. Res. 133(1):25 (2014)).

SUMMARY

In accordance with the invention, provided are methods and uses ofpeptide compounds having one or more activities for inhibiting cellproliferation, stimulating apoptosis or catastrophe, abrogating cellcycle G2 checkpoint of a cell; or treating undesirable cellproliferation or survival, such as that characterized by a cellproliferative disorder. For example, the invention provides methods anduses of inhibiting cell proliferation; abrogating cell cycle G2checkpoint of a cell; increasing sensitivity of a cell to a nucleic aciddamaging agent or treatment; increasing nucleic acid damage to a cell.

In one embodiment, a method or use for increasing nucleic acid damage ofa hyperproliferating cell or for the prophylaxis or treatment of a cellproliferative disorder in a mammal (e.g., a human) having a white bloodcell count within a normal range, includes administering a peptidecompound, wherein the peptide compound comprises any of the followingsequences: A) a peptide comprising residues denoted P1-P6, with thestructure, P1, P2, P3, P4, P5, P6 or P6, P5, P4, P3, P2, P1; wherein P1is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), an aminoacid that occupies a similar side chain space, or any amino acid withone or two aromatic, piperidine, pyrazine, pyrimidine, piperazine,morpholine or pyrimidine group(s), or one indole, pentalene, indene,naphthalene, benzofuran, benzothiophene, quinoline, indoline, chroman,quinoxaline, quinazoline group in the side chain; wherein P2 is Cha,Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, anamino acid that occupies a similar side chain space, or any amino acidwith one or two aromatic, piperidine, pyrazine, pyrimidine, piperazine,morpholine or pyrimidine group(s), or one indole, pentalene, indene,naphthalene, benzofuran, benzothiophene, quinoline, indoline, chroman,quinoxaline, or quinazoline group in the side chain; wherein P3, P4, P5are any amino acid, or wherein one or more of P3, P4, P5 is a simplecarbon chain such that the distance between P2 and P6 is about the sameas the distance when each of P3, P4, P5 are amino acids; wherein P6 isBpa, Phe4NO2, any one amino acid and Tyr, any one amino acid and Phe,any amino acid, or nothing; B) or the peptide of A), wherein the aminoacid having a simple carbon chain is 11-aminoundecanoic acid,10-aminodecanoic acid, 9-aminononanoic acid, 8-aminocaprylic acid,7-aminoheptanoic acid, 6-aminocaproic acid, or a similar structure withone or more unsaturated carbon bonds, and/or wherein the any one aminoacid is Ser, and/or wherein P4 is Trp, and/or wherein the amino acidthat occupies a similar side chain space is Tyr or Phe; or a peptidecomprising residues denoted P1-P12, with any the following structures:

P1, P2, P3, P4, P5, P6; P6, P5, P4, P3, P2, P1; P1, P2, P3, P4, P5, P6,P7, P8, P9, P10, P11, P12; P1, P2, P3, P4, P5, P6, P12, P11, P10, P9,P8, P7; P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12; P6, P5, P4,P3, P2, P1, P12, P11, P10, P9, P8, P7; P7, P8, P9, P10, P11, P12, P1,P2, P3, P4, P5, P6; P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1;P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6; P12, P11, P10, P9,P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P6, P9, P8, P7, P2, P1; P12,P11, P10, P6, P9, P4, P7, P2, P1; P1, P2, P7, P8, P9, P6, P11, P12; orP1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha, Nal(2),(Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, an amino acidthat occupies a similar side chain space (e.g. d- or 1-Tyr, d- or1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene, benzofuran, benzothiophene,quinoline, indoline, chroman, quinoxaline, or quinazoline group in theside chain; wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F),(Phe-4CF3), or an amino acid that occupies a similar side chain space,or any amino acid with one or two aromatic, piperidine, pyrazine,pyrimidine, piperazine, morpholine or pyrimidine group(s), or oneindole, pentalene, indene, naphthalene, benzofuran, benzothiophene,quinoline, indoline, chroman, quinoxaline, quinazoline group in the sidechain; wherein P3, P4, P5 are any amino acid, or wherein one or more ofP3, P4, P5 is a simple carbon chain such that the distance between P2and P6 is about the same as the distance when each of P3, P4, P5 areamino acids; wherein P6 is Bpa, Phe4NO2, any one amino acid and Tyr, anyone amino acid and Phe; and wherein at least three of P7, P8, P9, P10,P11, P12 are basic amino acids with the rest being any amino acid orabsent; or the peptide of C), wherein the amino acid having a simplecarbon chain is 11-aminoundecanoic acid, 10-aminodecanoic acid,9-aminononanoic acid, 8-aminocaprylic acid, 7-aminoheptanoic acid,6-aminocaproic acid, or a similar structure with one or more unsaturatedcarbon bonds, and/or, wherein the any one amino acid is Ser, and/or,wherein P4 is Trp and/or, wherein the amino acid that occupies a similarside chain space is Tyr or Phe; or a peptide comprising residues denotedP1-P12, with any the following structures:P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P12, P11, P10, P9,P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1;or P1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha, Nal(2),(Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, an amino acidthat occupies a similar side chain space, or any amino acid with one ortwo aromatic, piperidine, pyrazine, pyrimidine, piperazine, morpholineor pyrimidine group(s), or one indole, pentalene, indene, naphthalene,benzofuran, benzothiophene, quinoline, indoline, chroman, quinoxaline,or quinazoline group in the side chain; wherein P2 is Cha, Nal(2),(Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), an amino acid that occupiesa similar side chain space, or any amino acid with one or two aromatic,piperidine, pyrazine, pyrimidine, piperazine, morpholine or pyrimidinegroup(s), or one indole, pentalene, indene, naphthalene, benzofuran,benzothiophene, quinoline, indoline, chroman, quinoxaline, quinazolinegroup in the side chain; wherein P3, P4, P5 are any amino acid, orwherein one or more of P3, P4, P5 is a simple carbon chain such that thedistance between P2 and P6 is about the same as the distance when eachof P3, P4, P5 are amino acids; wherein P6 is Bpa, Phe4NO2, any one aminoacid and Tyr, any one amino acid and Phe, any amino acid, or nothing;and wherein at least three of P7, P8, P9, P10, P11, P12 are basic aminoacids with the rest being any amino acid or absent; or the peptide ofE), wherein the amino acid having a simple carbon chain isaminoundecanoic acid or 8-aminocaprylic acid, and/or, wherein the anyone amino acid is Ser, and/or, wherein the amino acid that occupies asimilar side chain space is Tyr or Phe; or a peptide comprising residuesdenoted P1-P12, with any the following structures: P1, P2, P3, P4, P5,P6 or P6, P5, P4, P3, P2, P1, wherein P1 is Cha, Nal(2),(Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr, or Phe;wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3),Bpa, Phe4NO2, Tyr, or Phe; wherein P3 is Ser, Arg, Cys, Pro, or Asn;wherein P4 is Trp; wherein P5 is Ser, Arg, or Asn; or wherein P3, P4, P5is a single aminoundecanoic acid or a single 8-aminocaprylic acid; andwherein P6 is Bpa, Phe4NO2, (Ser-Tyr), or (Ser-Phe); or

a peptide comprising residues denoted P1-P12, with any the followingstructures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P1, P2,P3, P4, P5, P6, P12, P11, P10, P9, P8, P7; P6, P5, P4, P3, P2, P1, P7,P8, P9, P10, P11, P12; P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8,P7; P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6; P7, P8, P9, P10,P11, P12, P6, P5, P4, P3, P2, P1; P12, P11, P10, P9, P8, P7, P1, P2, P3,P4, P5, P6; P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11,P6, P9, P8, P7, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; P1, P2,P7, P8, P9, P6, P11, P12; or P1, P2, P7, P4, P9, P6, P10, P11, P12;wherein P1 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3),Bpa, Phe4NO2, Tyr, or Phe; wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F),(Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr, or Phe; wherein P3 is Ser,Arg, Cys, Pro, or Asn; wherein P4 is Trp; wherein P5 is Ser, Arg, orAsn; or wherein P3, P4, P5 is a single aminoundecanoic acid or a single8-aminocaprylic acid; wherein P6 is Bpa, Phe4NO2, (d-Ser-d-Tyr), or(d-Ser-d-Phe); and wherein at least three of P7, P8, P9, P10, P11, P12are Arg or Lys with the rest being any amino acid or absent; or

a peptide comprising residues denoted P1-P12, with any the followingstructures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P12, P11,P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7,P2, P1; or P1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha, orNal(2); wherein P2 is (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3);wherein P3 is Ser; wherein P4 is Trp; wherein P5 is Ser or Asn; whereinP6 is Bpa, Phe4NO2, (Ser-Tyr), or (Ser-Phe); and wherein at least threeof P7, P8, P9, P10, P11, P12 are Arg with the rest being any amino acidor absent; or a peptide comprising residues denoted P1-P12, with any thefollowing structures: P1, P2, P3, P4, P5, P6 or P6, P5, P4, P3, P2, P1;wherein P1 is Cha, or Nal(2); wherein P2 is (Phe-2,3,4,5,6-F),(Phe-3,4,5F) or (Phe-4CF3); wherein P3 is Ser; wherein P4 is Trp;wherein P5 is Ser; and wherein P6 is Bpa, or (Ser-Tyr); or

a peptide comprising residues denoted P1-P12, with any the followingstructures: P1, P2, P3, P4, P5, P6; P6, P5, P4, P3, P2, P1; P1, P2, P3,P4, P5, P6, P7, P8, P9, P10, P11, P12; P1, P2, P3, P4, P5, P6, P12, P11,P10, P9, P8, P7; P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12; P6,P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7; P7, P8, P9, P10, P11,P12, P1, P2, P3, P4, P5, P6; P7, P8, P9, P10, P11, P12, P6, P5, P4, P3,P2, P1; P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6; P12, P11,P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P6, P9, P8, P7, P2,P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; P1, P2, P7, P8, P9, P6, P11,P12; or P1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha, orNal(2); wherein P2 is (Phe-2,3,4,5,6-F), (Phe-3,4,5F) or (Phe-4CF3);wherein P3 is any amino acid; wherein P4 is d- or 1-Trp; wherein P5 isany amino acid; wherein P6 is Bpa or (Ser-Tyr); wherein P7 is Arg;wherein P8 is Arg; wherein P9 is Arg; wherein P10 is Gln or Arg; whereinP11 is Arg; and wherein P12 is d- or 1-Arg, or

the peptide of K), wherein the any amino acid is Ser, or Pro; or

a peptide comprising residues denoted P1-P12, with any the followingstructures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P12, P11,P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7,P2, P1; or P1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha orNal(2); wherein P2 is (Phe-2,3,4,5,6-F); wherein P3 is Ser; wherein P4is Trp; wherein P5 is Ser; wherein P6 is Bpa or (Ser-Tyr); wherein P7 isArg; wherein P8 is Arg; wherein P9 is Arg; wherein P10 is Gln or Arg;wherein P11 is Arg; and wherein P12 is Arg; or

a prodrug thereof or a pharmaceutically acceptable salt thereof to themammal, thereby increasing nucleic acid damage of the hyperproliferatingcell or prophylaxis or treatment of the cell proliferative disorder.

In particular embodiments, a method or use employs a peptide compoundincluding any the following sequences: (d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg) (d-Arg) (d-Arg) (d-Gln)(d-Arg)(d-Arg);

(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F) (d-Cha);

(d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg) (d-Arg)(d-Gln) (d-Arg) (d-Arg) (d-Arg);

(d-Arg) (d-Arg) (d-Gln) (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F) (d-Cha);

(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Ser)(d-Trp)(d-Ser) (d-Bpa) (d-Arg)(d-Arg) (d-Arg) (d-Gln)(d-Arg) (d-Arg);

(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa);

(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg) (d-Arg);

(d-Arg) (d-Arg) (d-Gln) (d-Arg) (d-Arg) (d-Arg) (d-Cha)(d-Phe-2,3,4,5,6-F) (d-Ser)(d-Trp)(d-Ser) (d-Bpa);

(d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Cha)(d-Phe-2,3,4,5,6-F) (d-Ser) (d-Trp)(d-Ser)(d-Bpa);

(d-Cha)(d-Phe-2,3,4,5,6-F) (d-Ser) (d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg) (d-Arg) (d-Arg) (d-Arg);

(d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha);

(d-Bpa) (d-Ser) (d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg)(d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg);

(d-Arg)(d-Arg)(d-Bpa) (d-Arg) (d-Arg) (d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha);

(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Arg);

(d-Arg) (d-Arg) (d-Arg) (d-Bpa) (d-Arg)(d-Trp) (d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha);

(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg)(d-Trp) (d-Arg) (d-Bpa) (d-Arg)(d-Arg) (d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg) (d-Phe-2,3,4,5,6-F) (d-Cha); (d-Cha)(d-Phe-2,3,4,5,6-F) (d-Arg) (d-Trp)(d-Arg) (d-Bpa) (d-Arg) (d-Arg)(d-Arg) (d-Arg);

(d-Arg) (d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg) (d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha); or (d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg)(d-Arg) (d-Arg) (d-Bpa) (d-Arg) (d-Arg) (d-Arg).

In further particular embodiments, a method or use employs a peptidecompound including or consisting of any the following sequences:

(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:1);

(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg), or

(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha).

In additional particular embodiments, a method or use is practiced on amammal with a white blood cell count of less than about 11,000 whiteblood cells per microliter (wbc/μl) of blood; a mammal with a whiteblood cell count between about 4,000 to about 11,000 white blood cellsper microliter (wbc/μl) of blood; a mammal with a white blood cell countof less than about 10,000 white blood cells per microliter (wbc/μl) ofblood; a mammal with a white blood cell count of less than about 9,000white blood cells per microliter (wbc/μl) of blood; a mammal with awhite blood cell count between about 4,000 to about 9,000 white bloodcells per microliter (wbc/μl) of blood; a mammal with a white blood cellcount of less than about 8,000 white blood cells per microliter (wbc/μl)of blood; a mammal with a white blood cell count of less than about7,000 white blood cells per microliter (wbc/μl) of blood; or a mammalwith a white blood cell count of less than upper normal limit by eachclinical laboratories white blood cells per microliter (wbc/μl) ofblood.

Methods and uses include a peptide compound in a pharmaceuticalformulation. Invention methods and uses also include administration byany route. In particular embodiments, a peptide compound is administeredlocally, regionally or systemically.

Methods and uses include a pharmaceutically acceptable salt of a peptidecompound. In particular aspects, a pharmaceutically acceptable salt isany one or a combination of: acetate, sulfonate, sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogen-phosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methyl benzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate,methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, and mandelate.

Methods and uses include a peptide compound including or consisting of alength from 6 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40,40 to 50, 50 to 75, 75 to 100, 100 to 150, 150 to 200, or 200 to 300amino acid residues.

Methods and uses include a peptide compound including or consisting of acell penetrating molecule attached or conjugated thereto. In particularnon-limiting aspects, a cell penetrating molecule is joined to thepeptide compound by a covalent bond, or a peptide or a non-peptidelinker. In further particular non-limiting aspects, a cell penetratingpeptide comprises an alternating pattern of polar/charged amino acidsand non-polar, hydrophobic amino acids. In still further particularnon-limiting aspects, a cell penetrating peptide comprises apolycationic or amphipathic alpha-helix structure. In yet additionalparticular non-limiting aspects, cell penetrating peptide comprises apoly-Arginine (Arg) sequence (e.g., a peptide including or consisting of(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)).

In still further particular aspects, a peptide compound and/or the cellpenetrating peptide includes or consists of L- or D-isomer amino acids,or a mixture of L- and D-isomer amino acids.

In additional particular embodiments, a method or use further includesadministering a nucleic acid damaging agent, a nucleic acid damagingtreatment, an anti-proliferative agent, or an anti-proliferativetreatment. Non-limiting nucleic acid damaging agent, nucleic aciddamaging treatment, anti-proliferative agent, or anti-proliferativetreatment includes or consists of surgical resection, radiotherapy,ionizing or chemical radiation therapy, chemotherapy, immunotherapy,local or regional thermal (hyperthermia) therapy, vaccination, analkylating agent, an anti-metabolite, a plant extract, a plant alkaloid,nitrosourea, a hormone, or a nucleoside or nucleotide analogue.

In still further particular embodiments, a method or use furtherincludes or consists of the peptide compound administered prior to, withor after a nucleic acid damaging agent, a nucleic acid damagingtreatment, an anti-proliferative agent, or an anti-proliferativetreatment is administered. In particular aspects, a peptide compound isadministered less than 48 hours prior to or after a nucleic aciddamaging agent, a nucleic acid damaging treatment, an anti-proliferativeagent, or an anti-proliferative treatment is administered; a peptidecompound is administered less than 24 hours prior to or after a nucleicacid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or an anti-proliferative treatment isadministered; a peptide compound is administered less than 12 hoursprior to or after a nucleic acid damaging agent, a nucleic acid damagingtreatment, an anti-proliferative agent, or an anti-proliferativetreatment is administered; a peptide compound is administered less than6 hours prior to or after a nucleic acid damaging agent, a nucleic aciddamaging treatment, an anti-proliferative agent, or ananti-proliferative treatment is administered; a the peptide compound isadministered less than 4 hours prior to or after a nucleic acid damagingagent, a nucleic acid damaging treatment, an anti-proliferative agent,or an anti-proliferative treatment is administered; a peptide compoundis administered less than 2 hours prior to or after a nucleic aciddamaging agent, a nucleic acid damaging treatment, an anti-proliferativeagent, or an anti-proliferative treatment is administered; a peptidecompound is administered less than 1 hour prior to or after a nucleicacid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or an anti-proliferative treatment isadministered.

Non-limiting examples of a nucleic acid damaging agent oranti-proliferative agent include a drug. Non-limiting examples of anucleic acid damaging agent or anti-proliferative agent include aplatinum containing drug, such as cis-platin, carboplatin, nedaplatin,mitaplatin, satraplatin, picoplatin, triplatin, miriplatin, oroxaliplatin.

More particularly, methods and uses include or consist of administeringa platinum containing drug, cis-platin, carboplatin, oxaliplatin,pemetrexed, gemcitabine, 5-fiuorouracil (5-FU), rebeccamycin, adriamycin(ADR), bleomycin (Bleo), pepleomycin, cisplatin, cisplatinum, orcis-diamminedichloroplatinum(II) (CDDP), oxaliplatin, or camptotecin(CPT), cyclophosphamide, azathioprine, cyclosporin A, prednisolone,melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate,6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside,AZT, 5-azacytidine (5-AZC) or a 5-azacytidine related compound,actinomycin D, mithramycin, mitomycin C, carmustine, lomustine,semustine, streptozotocin, hydroxyurea, cisplatin, mitotane,procarbazine, dacarbazine, a taxane, vinblastine, vincristine,doxorubicin, dibromomannitol, radiation or a radioisotope. Particularnon-limiting examples of radiation include UVradiation, IR radiation,Xray, or alpha-, beta- or gamma-radiation. Particular non-limitingexamples of radioisotopes include I¹³¹, I¹²⁵, Sr⁸⁹, Sm¹⁵³, Y⁹⁰, orLu¹⁷⁷.

Invention methods and uses are applicable to a cell proliferative orhyperproliferative disorder or undesirable cell proliferation. Inparticular embodiments, a cell proliferative disorder comprises a tumoror cancer. In more particular embodiments, a cell proliferative disordercomprises a metastatic tumor or cancer.

Particular non-limiting examples of a tumor or cancer include a lungtumor or cancer, such as a small cell or non-small cell lung cancer, oran adenocarcinoma, squamous cell carcinoma or a large cell carcinoma.Further particular non-limiting examples of a tumor or cancer include acarcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma,melanoma, glioma, glioblastoma, meningioma, neuroblastoma,retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma,reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor,cancer or malignancy. Additional particular non-limiting examples oftumor or cancer is a lung, thyroid, head or neck, nasopharynx, throat,nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland,thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum,ileum, jejunum (small intestine), colon, rectum), genito-urinary tract(uterus, ovary, cervix, endometrial, bladder, testicle, penis,prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood,muscle, or skin neoplasia, tumor, or cancer. Still further particularnon-limiting examples of a tumor or cancer include a breast cancer,prostate cancer, pancreas cancer, gastric cancer, pleural mesothelioma,colon cancer, rectal cancer, large bowel cancer, small intestinalcancer, esophageal cancer, duodenal cancer, lingual cancer, pharyngealcancer, salivary gland cancer, cerebral tumor, schwanoma, liver cancer,kidney cancer, bile duct cancer, endometrial cancer, cervical cancer,uterine body cancer, ovarian cancer, bladder cancer, urethral cancer,skin cancer, angioma, malignant lymphoma, malignant melanoma, thyroidcancer, parathyroid cancer, nasal cancer, paranasal cancer, auditoryorgan cancer, carcinoma of oral floor, laryngeal cancer, parotid cancer,submandibular cancer, bone tumor, angiofibroma, retinal sarcoma, penilecancer, testicular tumor, pediatric solid cancer, Kaposi's sarcoma,tumor of maxillary sinus, fibrous histiocytoma, leiomyosarcoma,rhabdomyosarcoma, lymphoma, multiple myeloma or leukemia.

Particular non-limiting examples of a sarcoma include a lymphosarcoma,liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma,rhabdomyosarcoma or fibrosarcoma. Particular non-limiting examples of ahaematopoietic tumor, cancer or malignancy include a myeloma, lymphomaor leukemia.

Invention methods and uses include administering an amount of a peptidecompound effective to treat the tumor or cancer. In particular aspects,a method or use inhibits or reduces relapse, growth, progression,worsening or metastasis of the tumor or cancer; results in partial orcomplete destruction of the neoplastic, tumor, cancer or malignant cellmass, volume, size or numbers of cells, stimulating, inducing orincreasing neoplastic, tumor, cancer or malignant cell necrosis, lysisor apoptosis, reducing neoplasia, tumor, cancer or malignancy volumesize, cell mass, inhibiting or preventing progression or an increase inneoplasia, tumor, cancer or malignancy volume, mass, size or cellnumbers, or prolonging lifespan; results in reducing or decreasingseverity, duration or frequency of an adverse symptom or complicationassociated with or caused by the neoplasia, tumor, cancer or malignancy;or method results in reducing or decreasing pain, discomfort, nausea,weakness or lethargy, or results in increased energy, appetite, improvedmobility or psychological well being.

Moreover, provided are kits including peptide compounds optionally incombination with a nucleic acid damaging treatment (e.g., a nucleic aciddamaging agent), or an anti-proliferative agent. In one embodiment, akit includes a peptide compound and instructions for use in practicing amethod of the invention (e.g., administering to a mammal with a whiteblood cell count in a normal range, e.g., a mammal with a white bloodcell count of less than about 11,000 white blood cells per microliter(wbc/μl) of blood; a mammal with a white blood cell count between about4,000 to about 11,000 white blood cells per microliter (wbc/μl) ofblood; a mammal with a white blood cell count of less than about 10,000white blood cells per microliter (wbc/μl) of blood; a mammal with awhite blood cell count of less than about 9,000 white blood cells permicroliter (wbc/μl) of blood; a mammal with a white blood cell countbetween about 4,000 to about 9,000 white blood cells per microliter(wbc/μl) of blood; a mammal with a white blood cell count of less thanabout 8,000 white blood cells per microliter (wbc/μl) of blood; a mammalwith a white blood cell count of less than about 7,000 white blood cellsper microliter (wbc/μl) of blood; or a mammal with a white blood cellcount of less than upper normal limit by each clinical laboratorieswhite blood cells per microliter (wbc/μl) of blood).

DESCRIPTION OF DRAWINGS

FIG. 1 shows a dose response curve of each compound when used againstbleomycin treated Jurkat cells. X-axis indicates the dose and Y-axisindicates the % G2/M cells after treatment.

FIG. 2 shows a dose response curve of each compound when used againstcolchicine treated Jurkat cells. X-axis indicates the dose and Y-axisindicates the % G2/M cells after treatment.

FIGS. 3A and 3B Human pancreatic cancer derived cell line MIAPaCa2treated with (A) bleomycin (Bleo) or (B) adriamycin (ADR) with variousdoses of compounds. Harvested cells were stained for their DNA andanalyzed with flow cytometry. The % population of sub-G1 cells areindicated as dead cells.

FIGS. 4A to 4C are a schematic diagram of the structure activityrelationship of G2 checkpoint abrogator(1-Gly)(1-Arg)(1-Lys)(1-Lys)(1-Arg)(1-Arg)(1-Gln) (1-Arg)(1-Arg)(1-Cha)(1-Phe-2,3,4,5,6-F)(1-Arg)(1-Ser)(1-Pro)(1-Ser)(1-Tyr)(1-Tyr)(SEQ ID NO:78): (A) G2 checkpoint abrogation activity of amino acidsubstitutions for 1-Cha in bleomycin treated Jurkat cells are indictedin order,[1-Cha=1-Nal(2)]>[1-Ala(3-Bzt)=1-Nal(1)=1-Trp=1-Dph]>[1-Ala(tBu)=Cys(tBu)=Leu];(B) M phase checkpoint abrogating activity and/or non specific toxicityof amino acid substitutions for 1-Cha in cholchicine treated Jurkatcells in order, [Ala(3-Bzt)=1-Nal(1)=1-Dph]>[1-Cha=1-Nal(2)]; (C) G2checkpoint abrogating activity of amino acid substitution for1-Phe-2,3,4,5,6-F are indicted in order,1-(Phe-2,3,4,5,6-F)=1-(Phe-3,4,5-F)=1-(Phe-4CF3)]>[1-(Phe-3Br,4Cl,5Br)=1-(Phe-4Cl)=1-Tyr].

FIG. 5 shows G2 abrogating activity of various arginine rich sequences.Indicated peptides were added to Jurkat cells with or without bleomycin.The % G2/M cells is indicated on the Y-axis. X-axis is as follows: 1,Bleomycin alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and10, 50 μg/ml. Peptide sequences are as follows: rrrqrrkkr,

(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg)(d-Lys)(d-Lys)(d-Arg) (SEQ ID NO:79);CBP501, (d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg)(SEQ ID NO:80); no TAT,(d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F) (d-Cha) (SEQ ID NO:81);rqrr, (d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Gln)(d-Arg) (d-Arg)(SEQ ID NO:82); rrqrr, (d-Bpa)(d-Ser)(d-Trp) (d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:83); rrrq, (d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(SEQ ID NO:84); and rrrqr,(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg)(SEQ IDNO:85).

FIG. 6 shows G2 abrogating activity of various peptides without (d-Bpa).Indicated peptides were added to Jurkat cells with or without bleomycin.The % G2/M cells is indicated on the Y-axis. X-axis is as follows: 1,Bleomycin alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and10, 50 μl/ml. Peptide sequences are as follows: CBP0, (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg) (d-Arg)(SEQ ID NO:86); CBP451,(d-Tyr)(d-Ser)(d-Pro) (1-Trp)(1-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg)(SEQ ID NO:87); CBP452,(d-Tyr)(d-Ser)(1-Pro)(1-Trp)(1-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg) (d-Arg)(d-Gln) (d-Arg) (d-Arg)(SEQ ID NO:88); andCBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg)(d-Arg)(SEQ ID NO:80).

FIG. 7 shows G2 abrogating activity of various arginine rich and lysinerich peptide sequences. Indicated peptides were added to Jurkat cells asabove and the % G2/M cells calculated (Y-axis). Peptide sequences are asfollows: CBP603, (d-Bpa)(d-Ser) (d-Trp)(d-Ser)(d-Phe4NO2)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg)(d-Arg)(SEQ ID NO:89); CBP607, (d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg)(SEQID NO:90); CBP608, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg)(d-Arg)(SEQ ID NO91:); and CBP609, (d-Bpa)(d-Ser)(d-Trp) (d-Ser)(d-Phe-2,3,4,5,6-F) (d-Cha) (d-Lys) (d-Lys) (d-Lys) (d-Lys) (d-Lys)(d-Lys) (SEQ ID NO:92).

FIG. 8 shows that the location of the arginine rich portion of thesequence can be varied. Indicated peptides were added to Jurkat cells asabove and the % G2/M cells calculated (Y-axis). Peptide sequences are asfollows: CBP501, (d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg) (SEQ ID NO:80);CBP510, (d-Arg)(d-Arg) (d-Gln) (d-Arg) (d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F) (d-Ser)(d-Trp) (d-Ser) (d-Bpa) (SEQ IDNO:93); CBP511, (d-Arg)(d-Arg) (d-Gln) (d-Arg) (d-Arg)(d-Arg)(d-Bpa)(d-Ser) (d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:94);and CBP512, (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser) (d-Bpa) (SEQ ID NO:95).

FIG. 9 shows the structure of several studied substituted peptidesequences. G2 abrogating activity increased with the light shadedsubstitutions (*), M phase checkpoint abrogating activity and/or nonspecific toxicity increased with the darker shaded substitutions (**)and remained about the same for the rest of the substitutions.

FIG. 10 shows inhibition of tumor growth (human pancreatic carcinoma) inscid mice following treatment with CBP501 and cisplatin. Day0 indicatestreatment initiation. Mean tumor sizes with standard deviation for eachtreatment group are indicated on the Y-axis and the number of daysfollowing treatment initiation are indicated on the X-axis.

FIG. 11 shows G2 abrogating activity of peptides having a kinaseinhibiting sequence region and a sequence region based upon an HIV-TATtransduction sequence, as above. The % G2/M cells is indicated on theY-axis. X-axis is as follows: 1, Bleomycin alone; 2, 0.2 μg/ml; 3, 0.39μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8,12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml. Peptide sequences are asfollows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg) (SEQ ID NO:80); CBP700,(d-Arg)(d-Arg)(d-Bpa) (d-Arg) (d-Arg) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:96); CBP701, (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Trp) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:97);CBP702, (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Bpa) (d-Arg)(d-Trp) (d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:98); CBP703, (d-Arg)(d-Arg)(d-Arg)(d-Bpa) (d-Arg) (d-Arg) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:99).

FIG. 12 shows a comparison between G2 abrogating activity and Mabrogating activity and/or non specific toxicity of peptides withBleomycin for G2 abrogation analysis and colchicine for M abrogatingactivity and/or non specific toxicity. Indicated peptides were added toJurkat cells with bleomycin or colchicine. The % G2/M cells is indicatedon the Y-axis. X-axis is as follows: 1, Bleomycin or Colchicine alone;2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml.Peptide sequence is as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg).

FIG. 13 shows the molecular structure of CBP501,

(d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg) (d-Arg).

FIG. 14 shows Kaplan-Meyer analysis of overall survival in all treatedpatients in relation to baseline WBC: Kaplan-Meyer survival curves,Median OS and Hazard Ratio in relation to the baseline WBC in alltreated patients. The hazard ratio improves as the cut off leveldecreases and peaks at WBC 8000/μl as cut off level.

FIG. 15 shows Kaplan-Meyer analysis of overall survival in ICON enrolledpatients in relation to baseline WBC: Kaplan-Meyer survival curves,Median OS and Hazard Ratio in relation to the baseline WBC in ICONenrolled patients. The hazard ratio improves as the cut off leveldecreases and peaks at WBC 8000/μl as cut off level, and the differencebetween Arm A and Arm B was statistically significant at the peak.

FIG. 16 shows Kaplan-Meyer survival curves, median OS, patient numbers,hazard ratio and p values by Log-rank (Mantel-Cox) test in relation tothe WBC, >8000 or <8000, at screening in all treated population shown bythe arms.

FIG. 17 shows increased NET formation by activated neutrophils withCBP501 treatment.

FIG. 18 shows increased thrombin/anti-thrombin complexes by CBP501 invivo.

FIG. 19 shows CBP501 inhibited phagocytosis of both M1 and M2macrophages in vitro.

FIG. 20 shows suppression of TNF release from a mice macrophage cellline (RAW264.7).

DETAILED DESCRIPTION

The invention provides compounds including peptides and peptidomimeticsthat inhibit cell proliferation. The invention compounds are thereforeuseful for treating cell proliferative disorders or physiologicalconditions characterized by undesirable or unwanted cell proliferation,such as benign and malignant tumor cells. The ability of inventionpeptides and peptidomimetics to inhibit cell proliferation appears to bedue at least in part to abrogation of the cell cycle G2 checkpoint.Because cells can be induced to enter the cell cycle G2 checkpoint inresponse to nucleic acid damage to allow the cell to repair the damagebefore DNA replication and cell division occurs, by inhibiting the G2checkpoint, invention peptides and peptidomimetics sensitize cells tonucleic acid damaging agents and treatment protocols. Cells thataccumulate enough nucleic acid damage will be unable to complete repairof the damaged nucleic acid because the G2 checkpoint is disrupted. Suchcells will exhibit decreased proliferation (e.g., due to mutation of agene critical for survival that is not repaired) and eventually undergoapoptosis.

Cells having a normal G1 are less susceptible to accumulating damagednucleic acid since nucleic acid repair can also take place during G1.Thus, normal cells are less susceptible to the effects of the inventioncompounds. However, cells having an impaired or disrupted cell cycle G1checkpoint are more likely to accumulate damaged nucleic acid becausethe G1 checkpoint is impaired or disrupted making it less likely thatthe cells can completely repair the damaged nucleic acid. Thus, treatingG1 impaired or disrupted cells with an invention peptide orpeptidomimetic that disrupts the G2 checkpoint makes the cells even lesslikely to be able to complete repair of the damaged nucleic acid. G1impaired or disrupted cells are therefore particularly sensitive to suchinvention peptides and peptidomimetics. Thus, invention compoundsincluding peptides and peptidomimetics can be used to inhibit or preventcell proliferation in general and in particular inhibit proliferation ofcells having an impaired or disrupted G1 checkpoint.

Cells having an impaired or disrupted G1 cell cycle checkpoint includebut are not limited to cells that rapidly proliferate. Cellproliferative disorders and physiological conditions characterized byrapidly growing cells, undesirably growing cells or cells that surviveinstead of undergoing apoptosis frequently have impaired or disrupted G1cell cycle checkpoint. Thus, as it appears that the ability of inventionpeptides and peptidomimetics to inhibit proliferation or stimulateapoptosis is due, at least in part, to disrupting the G2 cell cyclecheckpoint, cells that rapidly or undesirably proliferate due to animpaired or disrupted G1 checkpoint are particularly attractive targets.

CBP501 is a cell cycle G2 checkpoint inhibiting peptide TAT-S216A(Suganuma, M., et al. Cancer Res. 59:5887 (1999)). A cell cyclephenotype-based screening method was employed to optimize TAT-S216A toreduce the accumulation of cancer cells in the cell cycle G2 phase inresponse to DNA damaging agents without affecting cell cycle phenotypeof normal cells (Sha, S., et al. Mol. Cancer Ther. 6:147 (2007)). CBP501was found to increase platinum concentration and platinum-DNA adductformation in CBP501-sensitive tumor cells and may operate,alternatively, or in addition to G2 checkpoint inhibition/disruption viaCalmodulin inhibition (Mine, N., et al. Mol. Cancer Ther. 10:1929(2011)).

Invention compounds including peptides and peptidomimetics may suppresscell proliferation by themselves without additional treatments thatdamage nucleic acid or that have anti-proliferative activity sincedisrupting G2 checkpoint will likely lead to the accumulation of nucleicacid damage as the cells divide. Accordingly, abnormal or undesirablyproliferating or surviving cells can be treated with a compound of theinvention alone, or in combination with a nucleic acid damagingtreatment (e.g., a chemical agent or treatment protocol), to inhibit orprevent proliferation of the cells or to stimulate cellapoptosis/catastrophe.

Unlike conventional anti-cell proliferative agents, which target rapidlyproliferating cells irrespective of whether the cells are normal orabnormal (e.g., cancer cell), invention compounds preferentially targetcells having an impaired or disrupted cell cycle G1 checkpoint. Forexample, CBP501, unlike cisplatin, does not affect the growth of HUVECcells (see, e.g., Table 3). CBP501 also does not affect M phase cellcycle arrest and/or non specific toxicity induced by colchicine (see,e.g., FIG. 12). Consequently, invention compounds are less likely toproduce excess undesirable side effects associated with conventionalanti-cell proliferative treatment agents, such as bone marrowsuppression, nausea, loss of appetite, diarrhea, and hair loss. Inaddition, because the vast majority of cancer cells have an impaired ordisrupted cell cycle G1 checkpoint, cancer cells will exhibit increasedsensitivity to invention compounds that abrogate cell cycle G2checkpoint. That normal cells are less susceptible also means thatinvention compounds including peptides and peptidomimetics can be usedin greater amounts.

In accordance with the invention, there are provided compounds includingpeptides and peptidomimetics having anti-cell proliferative activityand/or that abrogate the G2 cell cycle checkpoint. The peptides orpeptidomimetics include sequences that inhibit proliferation of a cellor that stimulate apoptosis of a cell. The peptides or peptidomimeticsalso include sequences that abrogate cell cycle G2 checkpoint. In oneembodiment, a contiguous peptide or peptidomimetic sequence includes thefollowing structure: P1, P2, P3, P4, P5, P6 (SEQ ID NO:1) or P6, P5, P4,P3, P2, P1 (SEQ ID NO:2); wherein P1 is d- or 1-Cha, d- or 1-Nal(2), d-or 1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), anamino acid that occupies a similar side chain space (e.g., d- or 1-Tyr,d- or 1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene group, benzofuran,benzothiophene, quinoline, indoline, chroman, quinoxaline, quinazolinegroup in the side chain; P2 is d- or 1-Cha, d- or 1-Nal(2), d- or1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or1-Bpa, d- or 1-Phe4NO2, an amino acid that occupies a similar side chainspace (e.g. d- or 1-Tyr, d- or 1-Phe), or any amino acid with one or twoaromatic, piperidine, pyrazine, pyrimidine, piperazine, morpholine orpyrimidine group(s), or one indole, pentalene, indene, naphthalene,benzofuran, benzothiophene, quinoline, indoline, chroman, quinoxaline,or quinazoline group in the side chain; P3, P4, P5 are any amino acid orone or more of P3, P4, P5 is a simple carbon chain such that thedistance between P2 and P6 is about the same as the distance when eachof P3, P4, P5 are amino acids (d- or 1-Trp is an example at P4; P6 is d-or 1-Bpa, d- or 1-Phe4NO2, any amino acid and d- or 1-Tyr (e.g.,d-Ser-d-Tyr), any amino acid and d- or 1-Phe (e.g., d-Ser-d-Phe), anyamino acid, or nothing. In various aspects, the amino acid having asimple carbon chain is d- or 1-11-aminoundecanoic acid, d- or1-10-aminodecanoic acid, d- or 1-9-aminononanoic acid, d- or1-8-aminocaprylic acid, d- or 1-7-aminoheptanoic acid, d- or1-6-aminocaproic acid, or a similar structure with one or moreunsaturated carbon bonds.

In another embodiment, a contiguous peptide or peptidomimetic sequenceincludes the following structure: P1, P2, P3, P4, P5, P6 (SEQ ID NO:3);P6, P5, P4, P3, P2, P1 (SEQ ID NO:4); P1, P2, P3, P4, P5, P6, P7, P8,P9, P10, P11, P12 (SEQ ID NO:5); P1, P2, P3, P4, P5, P6, P12, P11, P10,P9, P8, P7 (SEQ ID NO:6); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11,P12 (SEQ ID NO:7); P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7(SEQ ID NO:8); P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6 (SEQ IDNO:9); P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQ ID NO:10);P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6 (SEQ ID NO:11); P12,P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO:12); P12, P11,P6, P9, P8, P7, P2, P1 (SEQ ID NO:13); P12, P11, P10, P6, P9, P4, P7,P2, P1 (SEQ ID NO:14); P1, P2, P7, P8, P9, P6, P11, P12 (SEQ ID NO:15);or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:16); wherein P1 isd- or 1-Cha, d- or 1-Nal(2), d- or 1-(Phe-2,3,4,5,6-F), d- or1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or 1-Bpa, d- or 1-Phe4NO2, anamino acid that occupies a similar side chain space (e.g. d- or 1-Tyr,d- or 1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene, benzofuran, benzothiophene,quinoline, indoline, chroman, quinoxaline, or quinazoline group in theside chain; P2 is d- or 1-Cha, d- or 1-Nal(2), d- or1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), or anamino acid that occupies a similar side chain space (e.g. d- or 1-Tyr,d- or 1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene group, benzofuran,benzothiophene, quinoline, indoline, chroman, quinoxaline, quinazolinegroup(s) in the side chain; P3, P4, P5 are any amino acid or one or moreof P3, P4, P5 is a simple carbon chain such that the distance between P2and P6 is about the same as the distance when each of P3, P4, P5 areamino acids (d- or 1-Trp is an example at P4); P6 is d- or 1-Bpa, d- or1-Phe4NO2, any amino acid and d- or 1-Tyr (e.g., d-Ser-d-Tyr), any aminoacid and d- or 1-Phe (e.g., d-Ser-d-Phe), and at least three of P7, P8,P9, P10, P11, P12 are basic amino acids with the rest being any aminoacid or absent. In various aspects, the amino acid having a simplecarbon chain is d- or 1-11-aminoundecanoic acid, d- or1-10-aminodecanoic acid, d- or 1-9-aminononanoic acid, d- or1-8-aminocaprylic acid, d- or 1-7-aminoheptanoic acid, d- or1-6-aminocaproic acid or a a similar structure with one or moreunsaturated carbon bonds.

In a further embodiment, a contiguous peptide or peptidomimetic sequenceincludes the following structure: P1, P2, P3, P4, P5, P6, P7, P8, P9,P10, P11, P12 (SEQ ID NO:17); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3,P2, P1 (SEQ ID NO:18); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ IDNO:19); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:20); whereinP1 is d- or 1-Cha, d- or 1-Nal(2), d- or 1-(Phe-2,3,4,5,6-F), d- or1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or 1-Bpa, d- or 1-Phe4NO2, anamino acid that occupies a similar side chain space (e.g. d- or 1-Tyr,d- or 1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene, benzofuran, benzothiophene,quinoline, indoline, chroman, quinoxaline, or quinazoline group in theside chain; P2 is d- or 1-Cha, d- or 1-Nal(2), d- or1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), an aminoacid that occupies a similar side chain space (e.g. d- or 1-Tyr, d- or1-Phe), or any amino acid with one or two aromatic, piperidine,pyrazine, pyrimidine, piperazine, morpholine or pyrimidine group(s), orone indole, pentalene, indene, naphthalene, benzofuran, benzothiophene,quinoline, indoline, chroman, quinoxaline, quinazoline group in the sidechain; P3, P4, P5 are any amino acid or one or more of P3, P4, P5 is asimple carbon chain such that the distance between P2 and P6 is aboutthe same as the distance when each of P3, P4, P5 are amino acids (d- or1-Trp is an example at P4); P6 is d- or 1-Bpa, d- or 1-Phe4NO2, anyamino acid and d- or 1-Tyr (e.g., d-Ser-d-Tyr), any amino acid and d- or1-Phe (e.g., d-Ser-d-Phe), any amino acid, or nothing; and at leastthree of P7, P8, P9, P10, P11, P12 are basic amino acids with the restbeing any amino acid or absent. In various aspects, the amino acidhaving a simple carbon chain is d- or 1-aminoundecanoic acid or d- or1-8-aminocaprylic acid.

In yet another embodiment, a contiguous peptide or peptidomimeticsequence includes the following structure: P1, P2, P3, P4, P5, P6 (SEQID NO:21) or P6, P5, P4, P3, P2, P1 (SEQ ID NO:22); wherein P1 is d- or1-Cha, d- or 1-Nal(2), d- or 1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F),d- or 1-(Phe-4CF3), d- or 1-Bpa, d- or 1-Phe4NO2, d- or 1-Tyr, or d- or1-Phe; P2 is d- or 1-Cha, d- or 1-Nal(2), d- or 1-(Phe-2,3,4,5,6-F), d-or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or 1-Bpa, d- or 1-Phe4NO2, d-or 1-Tyr, or d- or 1-Phe; P3 is d- or 1-serine, d- or 1-arginine, d- or1-cysteine, d- or 1-proline, or d- or 1-asparagine; P4 is d- or1-tryptophan; and P5 is d- or 1-serine, d- or 1-arginine, or d- or1-asparagine; or P3, P4, P5 is a single d- or 1-aminoundecanoic acid ora single d- or 1-8-aminocaprylic acid; P6 is d- or 1-Bpa, d- or1-Phe4NO2, (d-Ser-d-Tyr), or (d-Ser-d-Phe).

In still another embodiment, a contiguous peptide or peptidomimeticsequence includes the following structure: P1, P2, P3, P4, P5, P6, P7,P8, P9, P10, P11, P12 (SEQ ID NO:23); P1, P2, P3, P4, P5, P6, P12, P11,P10, P9, P8, P7 (SEQ ID NO:24); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10,P11, P12 (SEQ ID NO:25); P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8,P7 (SEQ ID NO:26); P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6(SEQ ID NO:27); P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQID NO:28); P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6 (SEQ IDNO:29); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ IDNO:30); P12, P11, P6, P9, P8, P7, P2, P1 (SEQ ID NO:31); P12, P11, P10,P6, P9, P4, P7, P2, P1 (SEQ ID NO:32); P1, P2, P7, P8, P9, P6, P11, P12(SEQ ID NO:33); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:34);wherein P1 is d- or 1-Cha, Nal(2), d- or 1-(Phe-2,3,4,5,6-F), d- or1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or 1-Bpa, d- or 1-Phe4NO2, d- or1-Tyr, or d- or 1-Phe; P2 is d- or 1-Cha, d- or 1-Nal(2), d- or1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3), d- or1-Bpa, d- or 1-Phe4NO2, d- or 1-Tyr, or d- or 1-Phe; P3 is d- or1-serine, d- or 1-arginine, d- or 1-cysteine, d- or 1-proline, or d- or1-asparagine; P4 is d- or 1-tryptophan; P5 is d- or 1-serine, d- or1-arginine, or d- or 1-asparagine; or P3, P4, P5 is a single d- or1-aminoundecanoic acid or a single d- or 1-8-aminocaprylic acid; P6 isd- or 1-Bpa, d- or 1-Phe4NO2, (d-Ser-d-Tyr), or (d-Ser-d-Phe); and atleast three of P7, P8, P9, P10, P11, P12 are d- or 1-Arg or d- or 1-Lyswith the rest being any amino acid or absent.

In an additional embodiment, a contiguous peptide or peptidomimeticsequence includes the following structure: P1, P2, P3, P4, P5, P6, P7,P8, P9, P10, P11, P12 (SEQ ID NO:35); P12, P11, P10, P9, P8, P7, P6, P5,P4, P3, P2, P1 (SEQ ID NO:36); P12, P11, P10, P6, P9, P4, P7, P2, P1(SEQ ID NO:37); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:38);wherein P1 is d- or 1-Cha, or d- or 1-Nal(2); P2 is d- or1-(Phe-2,3,4,5,6-F), d- or 1-(Phe-3,4,5F), d- or 1-(Phe-4CF3); and atleast three of P7, P8, P9, P10, P11, P12 are d- or 1-Arg with the restbeing any amino acid or absent; P3 is d- or 1-serine; P4 is d- or1-tryptophan; P5 is d- or 1-serine or d- or 1-asparagine; P6 is d- or1-Bpa, d- or 1-Phe4NO2, (d- or 1-Ser-d- or 1-Tyr), or (d- or 1-Ser-d- or1-Phe).

In yet an additional embodiment, a contiguous peptide or peptidomimeticsequence includes the following structure: P1, P2, P3, P4, P5, P6 (SEQID NO:39) or P6, P5, P4, P3, P2, P1 (SEQ ID NO:40); wherein P1 is d- or1-Cha, or d- or 1-Nal(2); P2 is (d- or 1-Phe-2,3,4,5,6-F), (d- or1-Phe-3,4,5F) or (d- or 1-Phe-4CF3); P3 is d- or 1-Ser; P4 is d- or1-Trp; P5 is d- or 1-Ser; P6 is d- or 1-Bpa, or (d- or 1-Ser-d- or1-Tyr).

In a further embodiment, a contiguous peptide or peptidomimetic sequenceincludes the following structure: P1, P2, P3, P4, P5, P6 (SEQ ID NO:41);P6, P5, P4, P3, P2, P1 (SEQ ID NO:42); P1, P2, P3, P4, P5, P6, P7, P8,P9, P10, P11, P12 (SEQ ID NO:43); P1, P2, P3, P4, P5, P6, P12, P11, P10,P9, P8, P7 (SEQ ID NO:44); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11,P12 (SEQ ID NO:45); P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7(SEQ ID NO:46); P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6 (SEQID NO:47); P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQ IDNO:48); P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6 (SEQ IDNO:49); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ IDNO:50); P12, P11, P6, P9, P8, P7, P2, P1 (SEQ ID NO:51); P12, P11, P10,P6, P9, P4, P7, P2, P1 (SEQ ID NO:52); P1, P2, P7, P8, P9, P6, P11, P12(SEQ ID NO:53); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:54);wherein P1 is d- or 1-Cha, or d- or 1-Nal(2); P2 is (d- or1-Phe-2,3,4,5,6-F), (d- or 1-Phe-3,4,5F) or (d- or 1-Phe-4CF3); P3 isany amino acid (e.g., d- or 1-Ser, or d- or 1-Pro); P4 is d- or 1-Trp;P5 is any amino acid (e.g., d- or 1-Ser); P7 is d- or 1-Arg; P8 is d- or1-Arg; P9 is d- or 1-Arg; P10 is d- or 1-Gln or d- or 1-Arg; P11 is d-or 1-Arg; P12 is d- or 1-Arg; P6 is d- or 1-Bpa or (d- or 1-Ser-d- or1-Tyr).

In still another embodiment, a contiguous peptide or peptidomimeticsequence includes the following structure: P1, P2, P3, P4, P5, P6, P7,P8, P9, P10, P11, P12 (SEQ ID NO:55); P12, P11, P10, P9, P8, P7, P6, P5,P4, P3, P2, P1 (SEQ ID NO:56); P12, P11, P10, P6, P9, P4, P7, P2, P1(SEQ ID NO:57); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO:58);wherein P1 is d- or 1-Cha or d- or 1-Nal(2); P2 is (d- or1-Phe-2,3,4,5,6-F); P3 is d- or 1-Ser; P4 is d- or 1-Trp; P5 is d- or1-Ser; P7 is d- or 1-Arg; P8 is d- or 1-Arg; P9 is d- or 1-Arg; P10 isd- or 1-Gln or d- or 1-Arg; P11 is d- or 1-Arg; P12 is d- or 1-Arg; P6is d- or 1-Bpa or (d- or 1-Ser-d- or 1-Tyr).

In still further embodiments, a contiguous peptide or peptidomimeticsequence includes the following structure: (d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F) (d-Cha)(d-Arg) (d-Arg) (d-Arg) (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO:99); (d-Arg) (d-Arg) (d-Arg) (d-Gln)(d-Arg) (d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F) (d-Cha) (SEQ IDNO:100); (d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg) (d-Gln) (d-Arg) (d-Arg) (d-Arg) (SEQ ID NO:59); (d-Arg) (d-Arg)(d-Gln) (d-Arg) (d-Arg) (d-Arg) (d-Bpa) (d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F) (d-Cha) (SEQ ID NO:60); (d-Cha) (d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser) (d-Bpa) (d-Arg) (d-Arg) (d-Arg) (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO:61); (d-Arg) (d-Arg) (d-Arg) (d-Gln)(d-Arg) (d-Arg)(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Ser)(d-Trp)(d-Ser) (d-Bpa) (SEQ IDNO:62); (d-Cha) (d-Phe-2,3,4,5,6-F) (d-Ser) (d-Trp) (d-Ser) (d-Bpa)(d-Arg) (d-Arg) (d-Gln) (d-Arg) (d-Arg) (d-Arg) (SEQ ID NO:63); (d-Arg)(d-Arg) (d-Gln) (d-Arg) (d-Arg) (d-Arg) (d-Cha) (d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser) (d-Bpa) (SEQ ID NO:64); (d-Arg) (d-Arg) (d-Arg)(d-Arg) (d-Arg) (d-Arg) (d-Cha) (d-Phe-2,3,4,5,6-F) (d-Ser)(d-Trp)(d-Ser) (d-Bpa) (SEQ ID NO:65); (d-Cha) (d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser) (d-Bpa) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg)(d-Arg) (SEQ ID NO:66); (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Arg)(d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:67);(d-Bpa) (d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg) (d-Arg)(d-Arg) (d-Arg) (d-Arg) (d-Arg) (SEQ ID NO:68); (d-Arg)(d-Arg)(d-Bpa)(d-Arg) (d-Arg) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:69);(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Arg) (SEQ ID NO:70); (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Trp) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:71);(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg)(d-Trp) (d-Arg) (d-Bpa) (d-Arg)(d-Arg) (d-Arg) (SEQ ID NO:72); (d-Arg) (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Trp) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:73);(d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg)(d-Trp) (d-Arg) (d-Bpa) (d-Arg)(d-Arg) (d-Arg) (d-Arg) (SEQ ID NO:74); (d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg) (d-Arg) (d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:75); or (d-Cha) (d-Phe-2,3,4,5,6-F) (d-Arg) (d-Arg) (d-Arg)(d-Bpa) (d-Arg)(d-Arg)(d-Arg) (SEQ ID NO:76).

In still additional embodiments, a contiguous peptide or peptidomimeticsequence includes the following structure:

(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO:77).

Invention peptides and peptidomimetics optionally contain a poly-lysand/or arg sequence in order to assist traversing the cell membrane.Because other amino acid sequences (e.g., HIV tat, ligands for cellsurface receptors/proteins, etc.) are capable of traversing the membraneand other molecules can be used to facilitate cell entry of G2abrogating peptides and peptidomimetics (e.g., liposomes, micelles andother lipid molecules, viral and other vectors, electroporation, etc.),including poly-lys and/or poly-arg sequences is optional. Thus, inadditional embodiments, the peptides and peptidomimetics do not have apoly-lys and/or arg sequence that assists with cell entry. For example,in two particular embodiments, a minimum sequence without a poly-lys/argsequence assisting with cell membrane traversal includes P6, P5, P4, P3,P2, P1 e.g., d-Bpa, d-Ser, d-Trp, d-Ser, d-Phe-2,3,4,5,6F, d-Cha (SEQ IDNO:101); and d-Tyr, d-Ser, d-Pro, d-Trp, d-Ser, d-Phe-2,3,4,5,6F, d-Cha(SEQ ID NO:102). In two additional particular embodiments, a minimumsequence without a poly-lys/arg sequence assisting with cell membranetraversal includes, for example, d-Bpa, d-Cys, d-Trp, d-Ser,d-Phe-2,3,4,5,6F, d-Cha, d-Cys (SEQ ID NO:103); and d-Tyr, d-Cys, d-Pro,d-Trp, d-Ser, d-Phe-2,3,4,5,6F, d-Cha, d-Cys (SEQ ID NO:104); the Cysresidues are optionally cyclized.

As discussed, invention compounds have anti-cell proliferative activityor G2 abrogating activity alone. Anti-cell proliferative activity can beincreased by combining such invention compounds with treatments thatdirectly or indirectly cause nucleic acid damage. Anti-cellproliferative activity also can be increased by combining such inventioncompounds with treatments that inhibit cell proliferation whether or notthe treatments damage nucleic acid. The invention therefore furtherprovides compositions including a compound of the invention (e.g., apeptide or peptidomimetic sequence) and a nucleic acid damaging agent,and compositions including a compound of the invention (e.g., a peptideor peptidomimetic sequence) and an anti-proliferative agent.

As used herein, the terms “abrogate the cell cycle G2 checkpoint,”“disrupt the cell cycle G2 checkpoint,” “impair the cell cycle G2checkpoint” and grammatical variations thereof, means inhibiting a cellto arrest cell cycle at the G2 checkpoint. A cell in which the cellcycle G2 checkpoint is abrogated exhibits a decrease in the length oftime that the cell is in the G2 checkpoint, which can range from absenceof G2 checkpoint altogether to a G2 checkpoint having a decrease induration of minutes, hours, days, weeks or longer under appropriateconditions. Thus, a cell contacted with an invention compound has a G2checkpoint time shorter in length than the cell normally would have inthe absence of the compound. For example, a decrease in the length of G2checkpoint time would mean that a cell which is in G2 for a certaintime, e.g., 4 hours, when contacted with an invention compound, is in G2for less than 4 hours, e.g., 3.5, 3, 2.5, 2, 1 or fewer hours.

As used herein, the term “apoptosis” refers to programmed cell death,and associated changes in cell physiology, e.g., nucleic acidfragmentation, caspase activation, etc., as is understood in the art.The term “catastrophe” means cell death resulting from an error in themitotic process. In catastrophe, there are fewer features present thatare characteristic of apoptosis e.g., caspase activation, chromosomecondensation, etc.

As used herein, the terms “peptide,” “polypeptide” and “protein” areused interchangeably and refer to two or more amino acids covalentlylinked by an amide bond or non-amide equivalent. The peptides of theinvention can be of any length. For example, the peptides can have fromabout 5 to 100 or more residues, such as, 5 to 12, 12 to 15, 15 to 18,18 to 25, 25 to 50, 50 to 75, 75 to 100, or more in length. The peptidesof the invention include 1- and d-isomers, and combinations of 1- andd-isomers. The peptides can include modifications typically associatedwith post-translational processing of proteins, for example, cyclization(e.g., disulfide or amide bond), phosphorylation, glycosylation,carboxylation, ubiquitination, myristylation, or lipidation.

Peptides disclosed herein further include compounds having amino acidstructural and functional analogues, for example, peptidomimetics havingsynthetic or non-natural amino acids or amino acid analogues, so long asthe mimetic has one or more functions or activities. The compounds ofthe invention therefore include “mimetic” and “peptidomimetic” forms.

As used herein, the terms “mimetic” and “peptidomimetic” refer to asynthetic chemical compound which has substantially the same structuraland/or functional characteristics of the peptides of the invention. Themimetic can be entirely composed of synthetic, non-natural amino acidanalogues, or can be a chimeric molecule including one or more naturalpeptide amino acids and one or more non-natural amino acid analogs. Themimetic can also incorporate any number of natural amino acidconservative substitutions as long as such substitutions do not destroythe mimetic's activity. As with polypeptides of the invention which areconservative variants, routine testing can be used to determine whethera mimetic has the requisite activity, e.g., that it has detectable cellcycle G2 checkpoint abrogating activity. A mimetic, when administered toa subject or contacted on a cell, that detectably disrupts the G2 cellcycle checkpoint, would therefore have G2 checkpoint abrogatingactivity.

Peptide mimetic compositions can contain any combination of non-naturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., induce or stabilize a secondary structure, e.g., a betaturn, gamma turn, beta sheet, alpha helix conformation, and the like.For example, a polypeptide can be characterized as a mimetic when one ormore of the residues are joined by chemical means other than an amidebond. Individual peptidomimetic residues can be joined by amide bonds,non-natural and non-amide chemical bonds other chemical bonds orcoupling means including, for example, glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups alternative to the amide bond include, forexample, ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide and BackboneModifications,” Marcel Decker, N.Y.).

As discussed, a peptide can be characterized as a mimetic by containingone or more non-natural residues in place of a naturally occurring aminoacid residue. Non-natural residues are known in the art. Particularnon-limiting examples of non-natural residues useful as mimetics ofnatural amino acid residues are mimetics of aromatic amino acidsinclude, for example, D- or L-naphylalanine; D- or L-phenylglycine; D-or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- orL-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- orL-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D-or L-2-indole(alkyl)alanines; and D- or L-alkylainines, where alkyl canbe substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl,pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidicamino acid. Aromatic rings of a non-natural amino acid that can be usedin place a natural aromatic rings include, for example, thiazolyl,thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, andpyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution withnon-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; and sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) including, for example,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide Aspartyl or glutamylgroups can also be converted to asparaginyl and glutaminyl groups byreaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution, forexample, in addition to lysine and arginine, with the amino acidsornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues.

Arginine mimetics can be generated by reacting arginyl with one or morereagents including, for example, phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, or ninhydrin, optionally under alkalineconditions. Tyrosine residue mimetics can be generated by reactingtyrosyl with aromatic diazonium compounds or tetranitromethane.N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate.

Methionine mimetics can be generated by reaction with methioninesulfoxide. Proline mimetics of include, for example, pipecolic acid,thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline,3- or 4-methylproline, and 3,3,-dimethylproline. Histidine mimetics canbe generated by reacting histidyl with diethylprocarbonate orpara-bromophenacyl bromide. Other mimetics include, for example, thosegenerated by hydroxylation of proline and lysine; phosphorylation of thehydroxyl groups of seryl or threonyl residues; methylation of thealpha-amino groups of lysine, arginine and histidine; acetylation of theN-terminal amine; methylation of main chain amide residues orsubstitution with N-methyl amino acids; or amidation of C-terminalcarboxyl groups.

One or more residues can also be replaced by an amino acid (orpeptidomimetic residue) of the opposite chirality. Thus, any amino acidnaturally occurring in the L-configuration (which can also be referredto as R or S, depending upon the structure of the chemical entity) canbe replaced with the same amino acid or a mimetic, but of the oppositechirality, referred to as the D-amino acid, but which can additionallybe referred to as the R— or S— form.

Invention peptides and peptidomimetics further include modified forms ofthe sequences set forth herein, provided that the modified form retains,at least a part of, the function of the unmodified or reference peptideor peptidomimetic. For example, a modified peptide or peptidomimeticwill retain at least a part of cell proliferative inhibiting or G2abrogating activity, but may have increased or decreased cellproliferative inhibiting or G2 abrogating activity relative to referencepeptide or peptidomimetic.

Modified peptides and peptidomimetics can have one or more amino acidresidues substituted with another residue, added to the sequence ordeleted from the sequence. In one embodiment, the modified peptide orpeptidomimetic has one or more amino acid substitutions, additions ordeletions (e.g., 1-3, 3-5, 5-10 or more). In one aspect, thesubstitution is with an amino acid or mimetic whose side chain occupiesa similar space with the reference amino acid or mimetic (the amino acidor mimetic that is being substituted). In still another aspect, thesubstitution is with a non-human amino acid which is structurallysimilar to the human residue. In a particular aspect, the substitutionis a conservative amino acid substitution.

As used herein, the term “similar space” means a chemical moiety thatoccupies a three-dimensional space similar in size to a referencemoiety. Typically, a moiety that occupies a similar space will besimilar in size to the reference moiety. An amino acid or mimetic that“occupies a similar side chain space” has a side chain that occupies athree-dimensional space similar in size to the reference amino acid ormimetic. Specific examples for d-(Phe-2,3,4,5,6-F), 1-(Phe-2,3,4,5,6-F),d-(Phe-3,4,5F), 1-(Phe-3,4,5F), d-(Phe-4CF3) or 1-(Phe-4CF3), are (1 ord-Phe-2R1,3R2,4R3,5R4,6R5) where R1,R2,R3,R4,R5 can be chloride,bromide, fluoride, iodide, hydrogen, hydrogen oxide or absent. For smallmolecules, e.g., fluoride which has a size of about 1 Angstrom, similarspace may be absence of a moiety.

The term “conservative substitution” means the replacement of one aminoacid by a biologically, chemically or structurally similar residue.Biologically similar means that the substitution is compatible withbiological activity, e.g., anti-cell proliferative or G2 abrogatingactivity. Structurally similar means that the amino acids have sidechains with similar length, such as alanine, glycine and serine, orhaving similar size. Chemical similarity means that the residues havethe same charge or are both hydrophilic or hydrophobic. Particularexamples include the substitution of one hydrophobic residue, such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, serine for threonine, and the like.

Invention peptides and peptidomimetics therefore include peptides andpeptidomimetics having a sequence that is not identical to a sequence ofpeptides and peptidomimetics sequences set forth in Table 1. In oneembodiment, a peptide or peptidomimetic has a sequence having 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, or more identity with a sequence set forthin Table 1. In one aspect, the identity is over a defined area of thesequence, e.g., the amino or carboxy terminal 3-5 residues.

The compounds of the invention, including peptides and peptidomimeticscan be produced and isolated using any method known in the art. Peptidescan be synthesized, whole or in part, using chemical methods known inthe art (see, e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser.215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; and Banga,A. K., Therapeutic Peptides and Proteins, Formulation, Processing andDelivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.).Peptide synthesis can be performed using various solid-phase techniques(see, e.g., Roberge (1995) Science 269:202; Merrifield (1997) MethodsEnzymol. 289:3-13) and automated synthesis may be achieved, e.g., usingthe ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with themanufacturer's instructions.

Individual synthetic residues and polypeptides incorporating mimeticscan be synthesized using a variety of procedures and methodologies knownin the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, etal. (Eds) John Wiley & Sons, Inc., NY). Peptides and peptide mimeticscan also be synthesized using combinatorial methodologies. Techniquesfor generating peptide and peptidomimetic libraries are well known, andinclude, for example, multipin, tea bag, and split-couple-mix techniques(ses, for example, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curt Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; and Ostresh (1996) Methods Enzymol. 267:220-234). Modifiedpeptides can be further produced by chemical modification methods (see,for example, Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel(1995) Free Radic. Biol. Med. 19:373-380; and Blommers (1994)Biochemistry 33:7886-7896).

Peptides can also be synthesized and expressed as fusion proteins withone or more additional domains linked thereto for producing a moreimmunogenic peptide, to more readily isolate a recombinantly synthesizedpeptide, or to identify and isolate antibodies or antibody-expressing Bcells. Domains facilitating detection and purification include, forexample, metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals; protein A domains that allow purification on immobilizedimmunoglobulin; and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor Xa or enterokinase (Invitrogen,San Diego Calif.) between a purification domain and the peptide can beused to facilitate peptide purification. For example, an expressionvector can include a peptide-encoding nucleic acid sequence linked tosix histidine residues followed by a thioredoxin and an enterokinasecleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797;Dobeli (1998) Protein Expr. Purif. 12:404-14). The histidine residuesfacilitate detection and purification of the fusion protein while theenterokinase cleavage site provides a means for purifying the peptidefrom the remainder of the fusion protein. Technology pertaining tovectors encoding fusion proteins and application of fusion proteins isknown in the art (see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53).

The invention further provides nucleic acids encoding the peptides ofthe invention. In particular embodiments, a nucleic acid encodesinvention peptide sequences having a length of about 8 to 12, 12 to 15,15 to 18, 15 to 20, 18 to 25, 20 to 25, 25 to 35, 25 to 50 or 50 to 100amino acids or more in length.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to all forms of nucleic acid, including deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). The nucleic acids can be double,single strand, or triplex, linear or circular. Nucleic acids includegenomic DNA, cDNA, and antisense. RNA nucleic acid can be spliced orunspliced mRNA, rRNA, tRNA or antisense (e.g., RNAi). Nucleic acids ofthe invention include naturally occurring, synthetic, as well asnucleotide analogues and derivatives. Such altered or modifiedpolynucleotides include analogues that provide nuclease resistance, forexample. Nucleic acid lengths also can be less than the exemplifiedpeptide sequences. For example, a subsequence of any of the peptidesequences can encode a peptide having anti-proliferative or G2abrogating activity.

Nucleic acid can be produced using any of a variety of well knownstandard cloning and chemical synthesis methods and can be alteredintentionally by site-directed mutagenesis or other recombinanttechniques known to those skilled in the art. Purity of polynucleotidescan be determined through sequencing, gel electrophoresis and the like.

Nucleic acids of the invention may be inserted into a nucleic acidconstruct in which expression of the nucleic acid is influenced orregulated by an “expression control element,” the combination referredto as an “expression cassette.” The term “expression control element”means one or more sequence elements that regulates or influencesexpression of a nucleic acid sequence to which it is operatively linked.An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence.

The term “operatively linked” refers to a functional juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. Typically expression controlelements are juxtaposed at the 5′ or at the 3′ ends of the gene but canalso be intronic. Promoters are generally positioned 5′ of the codingsequence. A “promoter” is meant a minimal sequence element sufficient todirect transcription.

Expression control elements include promoters, enhancers, transcriptionterminators, gene silencers, a start codon (e.g., ATG) in front of aprotein-encoding gene. Expression control elements activate constitutivetranscription, inducible transcription (i.e., require an external signalfor activation), or derepress transcription (i.e., a signal turnstranscription off; removing the signal activates transcription).Expression cassettes can also include control elements sufficient torender gene expression controllable for specific cell-types or tissues(i.e., tissue-specific control elements).

Nucleic acids of the invention may be inserted into a plasmid forpropagation into a host cell and for subsequent genetic manipulation. Aplasmid is a nucleic acid that can be stably propagated in a host cell;plasmids optionally contain expression control elements in order todrive expression of the nucleic acid encoding peptide in the host cell.The term “vector” is used herein synonymously with a plasmid and mayalso include an expression control element for expression in a hostcell. Plasmids and vectors generally contain at least an origin ofreplication for propagation in a cell and a promoter. Plasmids andvectors are therefore useful for genetic manipulation of peptideencoding nucleic acids, for producing peptides, and for expressing thepeptides in host cells or whole organisms, for example.

Peptides may therefore be expressed in bacterial systems usingconstitutive promoters such as T7, or inducible promoters such as pL ofbacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter); in yeastsystems using constitutive promoters such as ADH or LEU2 or an induciblepromoter such as GAL (see, e.g., Ausubel et al., In: Current Protocolsin Molecular Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. &Wiley Interscience, 1988; Grant et al. Methods in Enzymology, 153:516(1987), eds. Wu & Grossman; Bitter Methods in Enzymology, 152:673(1987), eds. Berger & Kimmel, Acad. Press, N.Y.; and, Strathern et al.,The Molecular Biology of the Yeast Saccharomyces (1982) eds. Cold SpringHarbor Press, Vols. I and II; R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986); in insect cell systems using constitutive or inducible promoterssuch as ecdysone; and in mammalian cell systems using constitutivepromoters such as SV40, RSV, or inducible promoters derived from thegenome of mammalian cells such as metallothionein IIA promoter, heatshock promoter, or derived from mammalian virus such as adenovirus latepromoter or the inducible mouse mammary tumor virus long terminalrepeat. Peptide expression systems further include vectors designed forin vivo use including adenoviral vectors (U.S. Pat. Nos. 5,700,470 and5,731,172), adeno-associated vectors (U.S. Pat. No. 5,604,090), herpessimplex virus vectors (U.S. Pat. No. 5,501,979) and retroviral vectors(U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703 and WIPO publicationsWO92/05266 and WO92/14829). Bovine papilloma virus (BPV) has also beenemployed in gene therapy (U.S. Pat. No. 5,719,054). Such gene therapyvectors also include CMV based vectors (U.S. Pat. No. 5,561,063).

The invention therefore also provides nucleic acids encoding peptides ofthe invention inserted into host cells. In one embodiment, the host cellis a prokaryotic cell. In another embodiment, the host cell is aeukaryotic cell. In various aspects, the eukaryotic cell is a yeast ormammalian (e.g., human, primate, etc.) cell.

As used herein, a “host cell” is a cell into which a nucleic acid isintroduced that can be propagated, transcribed, or encoded peptideexpressed. The term also includes any progeny of the subject host cell.

Host cells include but are not limited to microorganisms such asbacteria or yeast; and plant, insect and mammalian cells. For example,bacteria transformed with recombinant bacteriophage nucleic acid,plasmid nucleic acid or cosmid nucleic acid expression vectors; yeasttransformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); or animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered forstable expression, are provided.

The expression vector also can contain a nucleic acid encoding aselectable marker conferring resistance to a selective pressure oridentifiable marker (e.g., β-galactosidase), thereby allowing cellshaving the vector to be identified, grown and expanded. Alternatively, aselectable marker can be on a second vector which is cotransfected intoa host cell with a first vector containing an invention polynucleotide.A number of selection systems may be used, including, but not limited tothe herpes simplex virus thymidine kinase gene (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase gene(Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and theadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells respectively.Antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, which confersresistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci.USA 78:2072 (1981)); the neomycin gene, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1(1981)); and the hygromycin gene, which confers resistance to hygromycin(Santerre et al., Gene 30:147 (1984)). Additional selectable genesinclude trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988));and ODC (ornithine decarboxylase), which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue (1987) In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory).

As used herein, the terms “nucleic acid damaging treatment” and “nucleicacid damaging agent” means any treatment regimen that directly orindirectly damages nucleic acid (e.g., DNA, cDNA, genomic DNA, mRNA,tRNA or rRNA). Specific examples of such agents include alkylatingagents, nitrosoureas, anti-metabolites, plant alkaloids, plant extractsand radioisotopes. Specific examples of agents also include nucleic aciddamaging drugs, for example, 5-fluorouracil (5-FU), capecitabine, S-1(Tegafur, 5-chloro-2,4-dihydroxypyridine and oxonic acid),5-ethynyluracil, arabinosyl cytosine (ara-C), 5-azacytidine (5-AC),2′,2′-difluoro-2′-deoxycytidine (dFdC), purine antimetabolites(mercaptopurine, azathiopurine, thioguanine), gemcitabine hydrochloride(Gemzar), pentostatin, allopurinol, 2-fluoro-arabinosyl-adenine(2F-ara-A), hydroxyurea, sulfur mustard (bischloroetyhylsulfide),mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide,thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, alkylsulfonate (busulfan), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU),procarbazine, decarbazine, rebeccamycin, anthracyclins such asdoxorubicin (adriamycin; ADR), daunorubibcin (Cerubicine), idarubicin(Idamycin) and epirubicin (Ellence), anthracyclin analogues such asmitoxantrone, actinimycin D, non intercalating topoisomerase inhibitorssuch as epipodophyllotoxins (etoposide=VP16, teniposide=VM-26),podophylotoxin, bleomycin (Bleo), pepleomycin, compounds that formadducts with nucleic acid including platinum derivatives (e.g.,cisplatin (CDDP), trans analogue of cisplatin, carboplatin, iproplatin,tetraplatin and oxaliplatin), camptothecin, topotecan, irinotecan(CPT-11), and SN-38. Specific examples of nucleic acid damagingtreatments include radiation (e.g., ultraviolet (UV), infrared (IR), oralpha-, beta- or gamma-radiation) and environmental shock (e.g.,hyperthermia).

As used herein, the terms “anti-proliferative treatment” and“anti-proliferative agent” means any treatment regimen that directly orindirectly inhibits proliferation of a cell, virus, bacteria or otherunicellular or multicellular organism regardless of whether or not thetreatment or agent damages nucleic acid. Particular examples ofanti-proliferative agents are anti-tumor and anti-viral drugs, whichinhibit cell proliferation or virus proliferation or replication.Specific examples include, inter alia, cyclophosphamide, azathioprine,cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine,busulphan, methotrexate, 6-mercaptopurine, thioguanine, cytosinearabinoside, taxol, vinblastine, vincristine, doxorubicin, actinomycinD, mithramycin, carmustine, lomustine, semustine, streptozotocin,hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine anddibromomannitol. Anti proliferative agents that cause nucleic acidreplication errors or inhibit nucleic acid replication such asnucleoside and nucleotide analogues (e.g., AZT or 5-AZC).

Invention peptides and peptidomimetics can also augment the anti-cellproliferative activity of microtubule stabilizing or destabilizingagents such as vinca alkaloids (vinblastine=VLB, vincristin=VCR,vinorelbine=VRLB, vinflunine=VFL), and taxanes (paclitaxel anddocetaxel=taxotare). Thus, such agents may be further included in thecompositions of the invention and used in the methods of the invention.

Cells that may be treated with the compounds of the invention includeany cell whose proliferation it is desired to inhibit or prevent invitro, ex vivo or in vivo. Particular target cells exhibit a shorterthan normal cell cycle G1 checkpoint time or have an impaired cell cycleG1 checkpoint such that the cells exit the G1 checkpoint before enoughtime has passed to complete nucleic acid repair. Candidate cellstherefore include cells that rapidly proliferate whether the cells arenormal or abnormal. Specific examples are benign or tumorous, metastaticor non-metastatic cells. Additional candidate cells can be identified bymeasuring their proliferation rate or the length of time that the cellsremain in G1 phase. Candidate cells can also be identified by contactinga test cell with an invention compound alone, or in combination with anucleic acid damaging treatment, and determining if the contacted cellexhibits decreased proliferation or increased cell death orapoptosis/catastrophe.

Invention compounds are therefore useful for inhibiting cellproliferation in vitro, ex vivo and in vivo. As such, subjects having orat risk of having a disorder or physiological condition characterized byabnormal or undesirable or unwanted cell proliferation or cell survival,or abnormal or deficient cell differentiation, can be treated with aninvention compound alone or in combination with a treatment thatdirectly or indirectly causes nucleic acid damage or ananti-proliferative treatment.

Thus, in accordance with the invention, there are provided methods forinhibiting cell proliferation, methods for increasing sensitivity of acell to a nucleic acid damaging agent or treatment and methods forincreasing nucleic acid damage to a cell in vitro, ex vivo and in vivo.In one embodiment, a method includes contacting a cell (e.g., a culturedcell or a cell present in a subject) with an amount of an inventionpeptide or peptidomimetic sufficient to inhibit proliferation of thecell. In another embodiment, a method includes contacting the cell withan amount of an invention peptide or peptidomimetic sufficient toincrease sensitivity of the cell to a nucleic acid damaging agent ortreatment. In yet another embodiment, a method includes contacting acell with an amount of an invention peptide or peptidomimetic sufficientto increase nucleic acid damage of the cell. In various aspects, amethod further includes contacting the cell with a nucleic acid damagingagent or exposing the cell to a nucleic acid damaging treatment.

Further provided are methods of treating a cell proliferative disorderor differentiative disorder in a subject, including conditionscharacterized by undesirable or unwanted cell proliferation or cellsurvival, conditions characterized by deficient or aberrant apoptosis,conditions characterized by aberrant or deficient cell survival, as wellas conditions characterized by aberrant or deficient celldifferentiation. In one embodiment, a method includes administering to asubject having or at risk of having a cell proliferative disorder, anamount of an invention peptide or peptidomimetic effective to treat thecell proliferative disorder. In one aspect, the amount is sufficient toimprove the subjects condition. In particular aspects, the improvementincludes, in at least a portion of the target cells (e.g., abnormallyproliferating cells), decreased cell proliferation, decreased numbers ofcells, inhibiting increases in the number of cells, increased apoptosis,or decreased survival. In yet another aspect, the subject isadministered an invention compound prior to, contemporaneously with, orafter administering a treatment that inhibits cell proliferation. Inadditional particular aspects, at least a part of the cells of the cellproliferative disorder are located in blood, breast, lung, thyroid, heador neck, brain, lymph, gastrointestinal tract, genito-urinary tract,kidney, pancreas, liver, bone, muscle, or skin.

In another embodiment, a method includes administering an amount ofcompound to the subject to treat a solid tumor. In yet anotherembodiment, a method includes administering an amount of compound to thesubject to treat a liquid tumor. In various aspects, the subject havingthe tumor is administered with an invention compound prior to,contemporaneously with, or after another anti-tumor therapy.

As used herein, the terms “proliferative disorder” and “proliferativecondition” mean any pathological or non-pathological physiologicalcondition characterized by aberrant or undesirable proliferation (e.g.,of a cell, virus, bacteria, fungus, etc.). The terms “cell proliferativedisorder” and “cell proliferative condition” mean any pathological ornon-pathological physiological condition characterized by aberrant orundesirable cell proliferation, as well as including conditionscharacterized by undesirable or unwanted cell proliferation or cellsurvival (e.g., due to deficient apoptosis), conditions characterized bydeficient or aberrant or deficient apoptosis, as well as conditionscharacterized by aberrant or undesirable or unwanted cell survival. Theterm “differentiative disorder” means any pathological ornon-pathological physiological condition characterized by aberrant ordeficient differentiation.

Proliferative or differentiative disorders amenable to treatment includediseases and non-pathological physiological conditions, both benign andneoplastic, characterized by abnormal or undesirable cell numbers, cellgrowth or cell survival. Such disorders or conditions may thereforeconstitute a disease state and include all types of cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, or may be non-pathologic, i.e., a deviationfrom normal but which is not typically associated with disease. Aspecific example of a non-pathologic condition that may be treated inaccordance with the invention is tissue re-growth from wound repair thatresults in scarring.

Cells comprising the proliferative or differentiative disorder may beaggregated in a cell mass or be dispersed. The term “solid tumor” refersto neoplasias or metastases that typically aggregate together and form amass. Particular examples include visceral tumors such as gastric orcolon cancer, hepatomas, venal carcinomas, lung and braintumors/cancers. A “liquid tumor” refers to neoplasias of thehaematopoetic system, such as lymphomas, myelomas and leukemias, orneoplasias that are diffuse in nature, as they do not typically form asolid mass. Particular examples of leukemias include acute and chroniclymphoblastic, myeolblasitc and multiple myeloma.

Such disorders include neoplasms or cancers, which can affect virtuallyany cell or tissue type, e.g., carcinoma, sarcoma, melanoma, metastaticdisorders or haematopoietic neoplastic disorders. A metastatic tumor canarise from a multitude of primary tumor types, including but not limitedto breast, lung, thyroid, head and neck, brain, lymphoid,gastrointestinal (mouth, esophagus, stomach, small intestine, colon,rectum), genito-urinary tract (uterus, ovary, cervix, bladder, testicle,penis, prostate), kidney, pancreas, liver, bone, muscle, skin, etc.

Carcinomas refer to malignancies of epithelial or endocrine tissue, andinclude respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. Exemplary carcinomas include those forming from thecervix, lung, prostate, breast, head and neck, colon, liver and ovary.The term also includes carcinosarcomas, e.g., which include malignanttumors composed of carcinomatous and sarcomatous tissues. Adenocarcinomaincludes a carcinoma of a glandular tissue, or in which the tumor formsa gland like structure.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,and fibrosarcoma.

As used herein, the term “haematopoietic proliferative disorder” means adisease involving hyperplastic/neoplastic cells of haematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. Typically, the diseases arise from poorlydifferentiated acute leukemias, e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia. Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML);lymphoid malignancies include, but are not limited to, acutelymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional malignant lymphomas include, but are not limited to,non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Treatments for use in combination with the invention compounds includeany anti-proliferative, nucleic acid damaging or anti-tumor treatment asdisclosed herein or known in the art. For example, an anti-cellproliferative or anti-tumor treatment may comprise radiation treatmentor surgical resection optionally in combination with drug treatment. Thetreatment may comprise administration of a chemical substance, such as aradioisotope, a drug, such as a chemotherapeutic agent, or genetictherapy, such as an anti-oncogene (e.g., Rb, DCC, p53, etc.), a dominantnegative oncogene or an antisense to an oncogene. The compounds can beadministered prior to, contemporaneously with or following othertreatment protocols. For example, a candidate subject for anti-cellproliferative therapy (e.g., radiation therapy, chemotherapy, genetherapy, surgical resection, etc.) can be administered an inventioncompound prior to initiating the anti-cell proliferative therapy. Thus,prophylactic treatment methods are provided.

The term “subject” refers to animals, typically mammalian animals, suchas primates (humans, apes, gibbons, chimpanzees, orangutans, macaques),domestic animals (dogs and cats), farm animals (horses, cattle, goats,sheep, pigs) and experimental animals (mouse, rat, rabbit, guinea pig).Subjects include animal disease models (e.g., tumor bearing mice).

Subjects appropriate for treatment include those currently undergoing orare candidates for treatment for a proliferative or differentiativedisorder or (e.g., anti-tumor therapy). Additional candidate subjectsinclude, for example, subjects at risk of developing a cellproliferative disorder. The invention methods are therefore applicableto treating a subject who is at risk of developing a cell proliferativedisorder but who has not yet exhibited overt symptoms of the disorder.At risk subjects can be identified as having a genetic predisposition orfamily history to developing a cell proliferative disorder. For example,subjects having an activated oncogene or having a mutation or deletionof a tumor suppressor gene are candidate subjects. At risk subjects cantherefore be identified using routine genetic screening for the presenceof the genetic lesion, or inquiry into the subjects' family history toestablish that they are at risk of the disorder. A particular example ofan at risk subject would be one with a family history or other geneticcharacteristic indicating predisposition to a cancer in which theneoplastic or drug-resistant neoplastic cells express CD40. A particularspecific example of a genetic disease is retinoblastoma, which is causedby a defect in the Rb tumor suppressor gene.

Amounts administered are typically in an “effective amount” or“sufficient amount” that is an amount sufficient to produce the desiredaffect. Effective amounts therefore include one or more of: decreasingcell proliferation, decreasing numbers of cells, inhibiting increasedproliferation, inhibiting increased numbers of cells, increasingapoptosis, or decreasing survival, of at least a portion of the cellscomprising the proliferating cells (e.g., at least some of the targetcells). Thus, for example, where it is desired to inhibit cellproliferation, an effective amount will be an amount that detectablydecreases cell proliferation or numbers of proliferating cells, orincreases cell apoptosis or decreases cell survival. The amount cantherefore be sufficient to reduce target cell numbers, stabilize targetcell numbers or inhibit increases in target cell numbers. For example,where the disorder comprises a solid tumor, reducing tumor size,stabilizing tumor size, or preventing further growth of the tumor, of atleast a portion of the tumor (e.g. inhibiting growth of 5-10% of thecells, or 10-20% or more of the cells comprising the tumor mass) is asatisfactory clinical endpoint. Where the disorder comprises a liquidtumor, reducing numbers of tumor cells, stabilizing tumor cell numbersor inhibiting further increases in tumor cell numbers, of at least asubpopulation of the tumor cells (e.g. inhibiting growth of 5-10% of thecells, or 10-20% or more of the cells) is a satisfactory clinicalendpoint.

In addition, amounts considered effective can prevent or inhibitprogression of the condition or disorder. For example, certain tumors asthey progress become increasingly aggressive, including progressing tometastatic forms. Thus, amounts also considered effective would resultin reducing or preventing the tumors from becoming increasinglyaggressive or from metastasizing. Accordingly, inhibiting or preventinga worsening of the disorder or condition, i.e., stabilizing thecondition is an additional satisfactory clinical endpoint.

Examination of a biological sample containing a liquid tumor (e.g.,blood or a tissue sample), can establish whether tumor cell mass ornumbers have been reduced, or inhibition of tumor cell proliferation hasoccurred. For a solid tumor, invasive and non-invasive imaging methodscan ascertain a reduction in tumor size, or inhibiting increases in thetumor size. Decreasing counts of receptor of a receptor positive tumor,can be used to assess reduction or inhibition of tumor cellproliferation. Amounts of hormone of a hormone producing tumor, e.g.,breast, testicular, or ovarian cancers, can be used to assess areduction or inhibition of proliferation of the tumor.

Effective amounts can also objectively or subjectively reduce ordecrease the severity or frequency of symptoms associated with thedisorder or condition. For example, an amount of an invention compoundthat reduces pain, nausea or other discomfort, or increases appetite orsubjective well being is a satisfactory clinical endpoint.

Effective amounts also include a reduction of the amount (e.g., dosage)or frequency of treatment with another protocol, which is considered asatisfactory clinical endpoint. For example, a cancer patient treatedwith an invention compound may require less nucleic acid damagingtreatment in order to inhibit cancer cell proliferation. In thisexample, an effective amount would include an amount that reduces thedosage frequency or amount of a nucleic acid damaging agent that thesubject is administered in comparison to the dosage frequency or amountadministered without treatment with a compound of the invention.

Methods of the invention that lead to an improvement in the subject'scondition or a therapeutic benefit may be relatively short in duration,e.g., the improvement may last several hours, days or weeks, or extendover a longer period of time, e.g., months or years. An effective amountneed not be a complete ablation of any or all symptoms of the conditionor disorder. Thus, a satisfactory clinical endpoint for an effectiveamount is achieved when there is a subjective or objective improvementin the subjects' condition as determined using any of the foregoingcriteria or other criteria known in the art appropriate for determiningthe status of the disorder or condition, over a short or long period oftime. An amount effective to provide one or more beneficial effects, asdescribed herein or known in the art, is referred to as an “improvement”of the subject's condition or “therapeutic benefit” to the subject.

An effective amount of an invention compound can be determined basedupon animal studies or optionally in human clinical trials. The skilledartisan will appreciate the various factors that may influence thedosage and timing required to treat a particular subject including, forexample, the general health, age, or gender of the subject, the severityor stage of the disorder or condition, previous treatments,susceptibility to undesirable side effects, clinical outcome desired andthe presence of other disorders or conditions. Such factors mayinfluence the dosage and timing required to provide an amount sufficientfor therapeutic benefit. The dosage regimen also takes intoconsideration the pharmacokinetics, i.e., the pharmaceuticalcomposition's rate of absorption, bioavailability, metabolism, andclearance (see, e.g., Egleton (1997) “Bioavailability and transport ofpeptides and peptide drugs into the brain” Peptides 18:1431-1439; andLanger (1990) Science 249:1527-1533). In addition, doses or treatmentprotocols may be specifically tailored to the subject or modified basedon pharmacogenomic data.

The compounds of the invention can therefore be administered alone or asa pharmaceutical composition, systemically, regionally (e.g., directedtowards an organ or tissue, e.g., by injection into the portal vein fortreating a cell proliferative disorder of the liver), or locally (e.g.,directly into a tumor mass), in accordance with any protocol or routethat achieves the desired effect. The compounds and pharmaceuticalcompositions can be administered as a single or multiple dose each day(e.g., at a low dose), or intermittently (e.g., every other day, once aweek, etc. at a higher dose). The compounds and pharmaceuticalcompositions can be administered via inhalation (e.g., intra-tracheal),orally, intravenously, intraarterially, intravascularly, intrathecally,intraperitonealy, intramuscularly, subcutaneously, intracavity,transdermally (e.g., topical), transmucosally (e.g., buccal, bladder,vaginal, uterine, rectal, or nasal), by multiple administrations,sustained release (e.g., gradual perfusion over time) or a single bolus.Implantable devices, including microfabricated devices, foradministering drugs are well known and are also applicable fordelivering compounds of the invention to a subject.

Compounds administered intravenously (IV) would be at about 0.01 mg/hrto about 1.0 mg/hr over several hours (typically 1, 3, or 6 hours),which can be repeated for one or more weeks with intermittent cycles.Considerably higher dosages (e.g., ranging up to about 10 mg/ml) can beused, particularly when the drug is administered to a secluded site andnot into the blood stream, such as into a body cavity or into a lumen ofan organ, e.g., the cerebrospinal fluid (CSF).

The invention therefore further provides pharmaceutical compositions.Such pharmaceutical compositions are useful for administration to asubject in vivo or ex vivo, and for treating a subject with theinvention compounds, for example.

As used herein the term “pharmaceutically acceptable” and“physiologically acceptable” includes solvents (aqueous or non-aqueous),solutions, emulsions, dispersion media, coatings, isotonic andabsorption promoting or delaying agents, compatible with pharmaceuticaladministration. A “pharmaceutical composition” or “pharmaceuticalformulation” therefore refers to a composition suitable forpharmaceutical use in a subject. The pharmaceutical compositions andformulations include an amount of an invention compound, for example, aneffective amount of a peptide or peptidomimetic, nucleic acid encodingsame, vector, or cell of the invention, and a pharmaceutically orphysiologically acceptable carrier.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration, systemic or local. Thus,pharmaceutical compositions include carriers, diluents, or excipientssuitable for administration by various routes.

Formulations or enteral (oral) administration can be contained in atablet (coated or uncoated), capsule (hard or soft), microsphere,emulsion, powder, granule, crystal, suspension, syrup or elixir.Conventional nontoxic solid carriers which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, can be used to prepare solid formulations. Supplementaryactive compounds (e.g., preservatives, antibacterial, antiviral andantifungal agents) can also be incorporated into the formulations. Aliquid formulation can also be used for enteral administration. Thecarrier can be selected from various oils including petroleum, animal,vegetable or synthetic, for example, peanut oil, soybean oil, mineraloil, sesame oil. Suitable pharmaceutical excipients include e.g.,starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol.

Pharmaceutical compositions for enteral, parenteral, or transmucosaldelivery include, for example, water, saline, phosphate buffered saline,Hank's solution, Ringer's solution, dextrose/saline, and glucosesolutions. The formulations can contain auxiliary substances toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents, detergents and the like. Additives canalso include additional active ingredients such as bactericidal agents,or stabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate or triethanolamine oleate. Additional parenteralformulations and methods are described in Bai (1997) J. Neuroimmunol.80:65-75; Warren (1997) J. Neurol. Sci. 152:31-38; and Tonegawa (1997)J. Exp. Med. 186:507-515. The parenteral preparation can be enclosed inampules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions for intradermal or subcutaneousadministration can include a sterile diluent, such as water, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid, glutathione orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

Pharmaceutical compositions for injection include aqueous solutions(where water soluble) or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride may be includedin the composition. The resulting solutions can be packaged for use asis, or lyophilized, the lyophilized preparation can later be combinedwith a sterile solution prior to administration.

Pharmaceutically acceptable carriers can contain a compound thatstabilizes, increases or delays absorption or clearance. Such compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; low molecular weight proteins; compositions that reduce theclearance or hydrolysis of peptides; or excipients or other stabilizersand/or buffers. Agents that delay absorption include, for example,aluminum monostearate and gelatin. Detergents can also be used tostabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers. To protectfrom digestion the compound can be complexed with a composition torender it resistant to acidic and enzymatic hydrolysis, or the compoundcan be complexed in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are known in theart (see, e.g., Fix (1996) Pharm Res. 13:1760-1764; Samanen (1996) J.Pharm. Pharmacol. 48:119-135; and U.S. Pat. No. 5,391,377, describinglipid compositions for oral delivery of therapeutic agents).

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be through nasal sprays orsuppositories (see, e.g., Sayani (1996) “Systemic delivery of peptidesand proteins across absorptive mucosae” Crit. Rev. Ther. Drug CarrierSyst. 13:85-184). For transdermal administration, the active compoundcan be formulated into ointments, salves, gels, or creams as generallyknown in the art. Transdermal delivery systems can also be achievedusing patches.

For inhalation delivery, the pharmaceutical formulation can beadministered in the form of an aerosol or mist. For aerosoladministration, the formulation can be supplied in finely divided formalong with a surfactant and propellant. In another embodiment, thedevice for delivering the formulation to respiratory tissue is in whichthe formulation vaporizes. Other delivery systems known in the artinclude dry powder aerosols, liquid delivery systems, inhalers, air jetnebulizers and propellant systems (see, e.g., Patton (1998)Biotechniques 16:141-143; Dura Pharmaceuticals, San Diego, Calif.;Aradigm, Hayward, Calif.; Aerogen, Santa Clara, Calif.; and InhaleTherapeutic Systems, San Carlos, Calif.).

Biodegradable, biocompatable polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations are known to those skilled in the art. The materials canalso be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells or tissues using antibodies or viral coat proteins)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known in the art, for example, asdescribed in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,522,811; 4,837,028;6,110,490; 6,096,716; 5,283,185; 5,279,833; Akimaru (1995) CytokinesMol. Ther. 1:197-210; Alving (1995) Immunol. Rev. 145:5-31; and Szoka(1980) Ann. Rev. Biophys. Bioeng. 9:467). Biodegradeable microspheres orcapsules or other biodegradeable polymer configurations capable ofsustained delivery of small molecules including peptides are known inthe art (see, e.g., Putney (1998) Nat. Biotechnol. 16:153-157).Compounds of the invention can be incorporated within micelles (see,e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23-28; Woodle (1992) Pharm.Res. 9:260-265). Peptides can be attached to the surface of the lipidmonolayer or bilayer. For example, peptides can be attached tohydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes(see, e.g., Zalipsky (1995) Bioconjug. Chem. 6:705-708). Alternatively,any form of lipid membrane, such as a planar lipid membrane or the cellmembrane of an intact cell, e.g., a red blood cell, can be used.Liposomal and lipid-containing formulations can be delivered by anymeans, including, for example, intravenous, transdermal (see, e.g.,Vutla (1996) J. Pharm. Sci. 85:5-8), transmucosal, or oraladministration.

A pharmaceutically acceptable formulation can incorporate about 1% to99.9% of active ingredient (e.g., peptide or peptidomimetic). Thepharmaceutical compositions can be sterilized by conventional,well-known sterilization techniques, or can be sterile filtered.

Additional pharmaceutical formulations and delivery systems are known inthe art and are applicable in the methods and compositions of theinvention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed.,Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles ofSolid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa.,(1993); and Poznansky et al., Drug Delivery Systems, R. L. Juliano, ed.,Oxford, N.Y. (1980), pp. 253-315)

The pharmaceutical formulations can be packaged in unit dosage form forease of administration and uniformity of dosage. “Unit dosage form” asused herein refers to physically discrete unitary dosages foradministration to the subject to be treated; each unit contains apredetermined quantity of compound that produces a desired effect incombination with a pharmaceutical carrier or excipient.

The following are abbreviations used herein:

Cha: cyclohexyl-alanine

Phe-2,3,4,5,6-F: Fluorides are at position 2,3,4,5,6, on Phenyl residueof Phenylalanine

F: Fluoride

Bpa: Benzoyl-phenylalanine

Nal(2): 2-Naphthyl-alanyl

Ala(3-Bzt): (3-Benzothienyl)-Alanine

Nal(1): 1-Naphthyl-alanyl

Dph: Diphenyl-Alanine

Ala(tBu): t-Butyl-alanyl

Cys(tBu): t-Butyl-cysteine

Phe-3,4,5-F: Fluorides are at position 3,4,5 on the Phenyl ofPhenylalanine

Phe-4CF3: CF3 is at position 4 on Phenyl residue of Phenylalanine

Phe-3Br,4Cl,5Br: Bromide is at position 3, Chloride is at position 4,and Bromide is at position 5 on the Phenyl of Phenylalanine

Phe-4Cl: Chloride is at position 4 on the Phenyl of Phenylalanine

P1, P2, P3, P4, P5, P6, etc., and (P1, P2, P3, P4, P5, P6, etc.); andP7, P8, P9, P10, P11, P12, etc., and (P7, P8, P9, P10, P11, P12, etc.):contiguous sequence of P1, P2, P3, P4, P5, P6, etc.; and P7, P8, P9,P10, P11, P12, respectively.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

As used herein, the singular forms “a”, “and,” “the” and “is” includeplural referents unless the context clearly indicates otherwise. Thus,for example, reference to a “compound” includes a plurality of compoundsand reference to “a residue” or an “amino acid” includes reference toone or more residues and amino acids.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

This example describes materials and several methods. This example alsodescribes the sequences of analyzed peptides/peptidomimetics.

Chemicals and Reagents Bleomycin was purchased from Wako Pure ChemicalCo. (Osaka, Japan) and it was dissolved in distilled H₂O to 10 mg/ml.Propidium iodide (PI) and adriamycin were purchased from Sigma (St.Louis, Mo.).

Cell Culture A human T-cell leukemia-derived cell line, Jurkat, wascultured in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum(IBL: Immuno-Biological Laboratories, Gunma, Japan) at 37° C./5% CO₂.Human pancreatic cancer derived cell line, MIAPaCa2 was cultured in DMEMwith 10% fetal calf serum at 37° C./5% CO₂.

Cell-Cycle Analysis The cell cycle status of the cells treated withbleomycin or adriamcin were analyzed by flow cytometry as described byKawabe (1997) Nature 385:454-458. In brief, two million cells werere-suspended and incubated in 200 μl Krishan's solution (0.1% Sodiumcitrate, 50 μg/ml PI, 20 μg/ml RNase A and 0.5% NP-40) for 1 hr at 4° C.and analyzed by a flow cytometry, FACScan™ (Beckton Dickinson, MountainView, Calif.) with the program CELLQuest™ (Beckton Dickinson).

TABLE 1 Sequences and Corresponding Code Names of exemplary peptides/peptidomimetics.(l-Tyr)(l-Gly)(l-Arg)(l-Lys)(l-Lys)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Arg)(l-Cha)BP413(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)(l-Pro)(l-Ser)(l-Tyr)(l-Tyr) (SEQ ID NO: 105)(l-Tyr)(l-Gly)(l-Arg)(l-Lys)(l-Lys)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Arg)(l-Cha)BP420(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)(l-Pro)(l-Ser)(l-Tyr) (SEQ ID NO: 106)(l-Arg)(l-Arg)(l-Arg)(l-Cha)(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)(l-Pro)(l-Ser)(l-Tyr)BP430 (l-Tyr) (SEQ ID NO: 107)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Arg)(l-Cha)(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)BP431 (l-Pro)(l-Ser)(l-Tyr)(l-Tyr) (SEQ ID NO: 108)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Arg)(l-Cha)(l-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)BP432 (l-Pro)(l-Ser)(l-Tyr) (SEQ ID NO: 109)(l-Tyr)(l-Gly)(l-Arg)(l-Lys)(l-Lys)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Arg)(l-Cha)BP440(l-Phe-2,3,4,5,6-F)(l-aminoundecanoic acid)(l-Tyr)(l-Tyr) (SEQ ID NO: 110)(d-Tyr)(d-Tyr)(d-Ser)(l-Gly)(d-Ser)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)BP450(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Lys)(d-Lys)(d-Arg)(l-Gly)(d-Tyr) (SEQ ID NO: 111)(d-Tyr)(d-Ser)(d-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP451 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 87)(d-Tyr)(d-Ser)(l-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP452 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 88)(d-Tyr)(d-Ser)(d-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Pro)(d-Arg)(d-Arg)(d-Arg)BP454 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 112)(d-Tyr)(d-Ser)(l-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(l-Pro)(d-Arg)(d-Arg)(d-Arg)BP455 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 113)(l-Tyr)(l-Tyr)(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP460(d-Gln)(d-Arg)(d-Arg)(d-Lys)(d-Lys)(d-Arg)(l-Gly)(d-Tyr) (SEQ ID NO: 114)(l-Tyr)(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)BP461 (d-Arg)(d-Arg)(d-Lys)(d-Lys)(d-Arg)(l-Gly)(d-Tyr) (SEQ ID NO: 115)(l-Tyr)(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 116)BP462(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)BP463 (d-Arg)(d-Lys)(d-Lys)(d-Arg)(l-Gly)(d-Tyr) (SEQ ID NO: 117)(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 118)BP464(l-aminoundecanoic acid)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)BP465 (d-Arg) (SEQ ID NO: 119)(l-8-aminocaprylic acid)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)BP466 (d-Arg) (SEQ ID NO: 120)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 121) BP470(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 122)BP471(d-Tyr)(d-Ser)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP481 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 123)(d-Tyr)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP500 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 124)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)BP501 (d-Arg)(d-Arg) (SEQ ID NO: 80)(d-Bpa)(l-8-aminocaprylic acid)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Gln)BP502 (d-Arg)(d-Arg) (SEQ ID NO: 125)(d-Bpa)(l-8-aminocaprylic acid)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)BP503 (d-Arg)(d-Arg) (SEQ ID NO: 126)(d-Asp)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP504 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 127)(d-Bpa)(d-Asp)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP505 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 128)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Asp)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)BP506 (d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 129)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)BP510 (d-Ser)(d-Bpa) (SEQ ID NO: 93)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)BP511 (d-Cha) (SEQ ID NO: 94)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)BP512 (d-Bpa) (SEQ ID NO: 95)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)BP601 (SEQ ID NO: 130)(d-Bpa)(l-8-aminocaprylic acid)(d-Bpa)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)BP602 (SEQ ID NO: 131)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe4No2)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)BP603 (SEQ ID NO: 89)(d-Bpa)(d-Pro)(d-Trp)(d-Pro)(d-Phe4NO2)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)BP604 (SEQ ID NO: 132)(d-Bpa)(d-Pro)(d-Trp)(d-Pro)(d-Phe4NO2)(d-Nal2)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)BP605 (SEQ ID NO: 133)(d-Phe4NO2)(d-Pro)(d-Trp)(d-Pro)(d-Phe4NO2)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)BP606 (d-Arg) (SEQ ID NO: 134)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)BP607 (SEQ ID NO: 90)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)BP608 (d-Arg) (SEQ ID NO: 91)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Lys)(d-Lys)(d-Lys)(d-Lys)(d-Lys)BP609 (d-Lys) (SEQ ID NO: 92)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 96)BP700(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 97)BP701(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)BP702 (SEQ ID NO: 98)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 99)BP703(d-Bpa)(d-Cys)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6F)(d-Cha)(d-Cys) (SEQ ID NO: 135)BP524(d-Tyr)(d-Cys)(d-Pro)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6F)(d-Cha)(d-Cys) (SEQ ID NO: 136)BP721

Example 2

This example describes data indicating the G2 abrogating activity ofvarious peptides, and the effect of various sequence permutations onactivity including the effect of decreasing sequence length.

Flow cytometry analysis of G2 checkpoint abrogation was performed usinghuman leukemia derived Jurkat cell line. In brief, cultured cells weretreated with various doses of peptide/peptidomimetic and 40 μg/mlbleomycine for 24 hr. The DNA of the cells was stained with propidiumiodide and analyzed by flow cytometry. These results are summarized inTable 2.

A dose response curve of each peptide/peptidomimetic when used againstbleomycin treated Jurkat cells are shown in FIGS. 1, 5, 6, 7, 8, 11 and12; the Y-axis indicates the % G2/M Jurkat cells 24 hrs after thetreatment.

Flow cytometry analysis of M phase checkpoint abrogation by thecompounds was performed using human T cell leukemia Jurkat cell linetreated with colchicine (5 μg/ml or 0.5 μg/ml) and various doses ofpeptide/peptidomimetics for 24 hr (FIG. 12). The DNA of the cells wasstained and analyzed by flow cytometry as described above. These resultsare also summarized in Table 2.

Dose response curves of each peptide/peptidomimetic when used againstcolchicine treated Jurkat cells are shown in FIGS. 2 and 14; the Y-axisindicates the % G2/M Jurkat cells 24 hrs after the treatment.

TABLE 2 Doses of compounds that induce G2 checkpoint abrogation or sideeffect Appearance of side Appearance of side G2 abrogating effect whenused with Code effect when used alone dose Colchicine name (μM) (μM)(μM) CBP441 >50 >50 >50 CBP462 >50 >50 >50 CBP464 >50 >50 >50CBP470 >50 >50 >50 CBP430 >50 50 >50 CBP481 >50 >6.25 >12.5 CBP431 >50≧3.125 >50 CBP420 >50 ≧1.56 ≧50 CBP440 >12.5 ≧1.56 >3.125 CBP413 >25≧1.56 >25 CBP450 >6.25 ≧0.78 >6.25 CBP460 >3.125 ≧0.39 >3.125CBP461 >6.25 ≧0.39 >6.25 CBP463 >6.25 ≧0.39 >6.25 CBP500 >50 ≧0.39 >12.5CBP501 >50 ≧0.39 >25

The “Appearance of side effect when used alone” indicates thepeptide/peptidomimetic dose that produced Jurkat cell cycle disturbance,i.e., the appearance of significant amounts of SubG1 cells (dead cells)or cells in which the DNA content of each varies more than usual. Forexample, G1 cells usually exhibit a sharp peak in FACS analysis, butfollowing treatment the peak becomes broader and lower when the cellcycle is disturbed indicating improper cell cycle progression or thebeginning of cell death. The “G2 abrogating dose” indicates thepeptide/peptidomimetic dose with 40 μg/ml bleomycine that produceddetectable G2 checkpoint abrogation activity following treatment for 24hours. The “Appearance of side effect when used with colchicine”indicates the peptide/peptidomimetic dose with 5 μg/ml colchicine thatproduced Jurkat cell cycle disturbance following treatment for 24 hours.

The G2 checkpoint abrogating activity of CBP501 when combined withcis-platin was studied in various cells lines. Briefly, cis-platin (3μg/ml) and CBP501 (0.4, 2 and 10 μM) were simultaneously added to thecell culture which was incubated 3 hr at 37 degree with 5% CO₂. Themedium was aspirated, fresh medium without these compounds was added andthe cells were incubated for an additional 45 hr. The cells includingfloating cells were harvested using trypsin-EDTA solution, incubatedwith Krishan's solution and analyzed for DNA content by flow cytometryas previously described. These results are summarized in Table 3. Shadedhighlighting, other than HUVEC, denote cell lines having a significantloss of G2 population and increased subG1 population, indicating G2checkpoint abrogation and sensitization to cisplatin by CBP501. Theobservation that HUVEC cells, which are cells having a normal G1checkpoint, were not sensitized, at least up to 50 μM CBP501, indicatesthat CBP501 is specific for the G2 checkpoint rather than non specific.

TABLE 3 G2 checkpoint abrogating doses of CBP501 against various celllines.

The G2 checkpoint abrogating activity of various compounds at differentdoses on human pancreatic cancer derived cell line MIAPaCa2 treated withbleomycine (Bleo) or adriamycin (ADR) was studied. Briefly, cells wereincubated with the compounds and bleomycine (10 μg/ml) or adriamycin (1μg/ml) for 3 hours. The medium was changed and incubated for anadditional 21 hours. Harvested cells were stained for DNA by propiumiodide and analyzed with flow cytometry as previously described. The %of the sub-G1 cell population is indicated as dead cells in FIG. 3. Theresults indicate that CBP501 sensitized MIAPaCa2 cells to both bleomycinand adriamycin in a dose dependent manner.

FIGS. 4A and 4C are a summary of the G2 checkpoint abrogating activityperformed with pairs of peptides in which one amino acid residue isdifferent from the other. The G2 checkpoint abrogating activity of thesepeptides was analyzed using bleomycin treated Jurkat cells as describedabove. FIG. 4B is a summary of M checkpoint abrogating activity and/ornon specific toxicity analysis performed with pairs of peptides in whichone amino acid residue is different from the other. The M checkpointabrogating activity and/or non specific toxicity of these peptides wasanalyzed using colchicine treated Jurkat cells as described above.

The G2 checkpoint abrogating activity of various arginine rich sequencesat different doses on cells treated with bleomycine was studied.Briefly, peptides were added to culture medium of Jurkat cells withbleomycin (40 μg/ml) at 0.2 μg/ml, 0.39 μg/ml, 0.78 μg/ml, 1.56 μg/ml,3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml. Cells weresubsequently harvested after 24 hours, stained with Krishan's solution,and analyzed with flow cytometry as previously described. The % G2/Mcells (Y-axis) was plotted against the peptide doses (X-axis) in FIG. 5.The data indicate that the “(d-Arg) (d-Arg) (d-Arg)(d-Gln) (d-Arg)(d-Arg) (SEQ ID NO:137)” basic residue rich sequence is the bestsequence compared to sequences having fewer or greater numbers ofresidues.

The G2 checkpoint abrogating activity of peptides without (D-Bpa) atdifferent doses on cells treated with bleomycine was studied. Briefly,peptides were added to culture medium of Jurkat cells with bleomycin (40μg/ml) at 0.2 μg/ml, 0.39 μg/ml, 0.78 μg/ml, 1.56 μg/ml, 3.125 μg/ml,6.25 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml. Cells were subsequentlyharvested and analyzed with flow cytometry as previously described. The% G2/M cells (Y-axis) was plotted against the peptide doses (X-axis) inFIG. 6. This result indicates that the sequence(Tyr)(Ser)(Pro)(Trp)(Ser) (Phe-2,3,4,5,6F)(Cha) (SEQ ID NO:138) hascomparable G2 checkpoint abrogating activity to the sequence(Bpa)(Ser)(Trp)(Ser)(Phe-2,3,4,5,6F)(Cha) (SEQ ID NO:139).

The G2 checkpoint abrogating activity of arginine rich and lysine richsequences at different doses on cells treated with bleomycine wasstudied. Briefly, peptides were added to culture medium of Jurkat cellswith bleomycin (40 μg/ml) at the indicated dose (X-axis). Cells weresubsequently harvested and analyzed with flow cytometry as previouslydescribed. The % G2/M cells (Y-axis) was plotted against the peptidedoses in FIG. 7. The results indicate that Arg sequences appear toprovide better activity than Lys sequences for the basic amino acid richsequence and that Gln is not essential for function of the sequence.

The G2 checkpoint abrogating activity of sequences in which the locationof the arginine rich region is varied was studied. Briefly, peptideswere added to culture medium of Jurkat cells with bleomycin (40 μg/ml)at the indicated dose (X-axis) for 24 hours. Cells were subsequentlyharvested and analyzed with flow cytometry as previously described. The% G2/M cells (Y-axis) was plotted against the peptide doses in FIG. 8.

The data indicate that the G2 abrogating activity of the peptides is notsignificantly altered by changing the location of the arginine richregion. In addition, CBP501 was soluble in water, whereas CBP511 wasnot. This difference can be advantageous for particular drug deliverysystems, since some systems prefer water insoluble compounds.

FIG. 9 illustrates a summary of the analysis performed with variouspeptide pairs in which only one amino acid residue was different betweenthe pairs. The G2 checkpoint abrogating activity of these peptides wasanalyzed using bleomycin treated Jurkat cells as described.

The size, charge and hydrophobicity of each amino acid determine howeffectively the sequence fits into a target molecule. The side chain ofthe peptide or peptidomimetic would move freely, so even with one or twounfavorable side chains the peptide or peptidomimetic could fit a pocketor groove of the target molecule. The summary indicates that there arepreferable sizes for each side chain which suggest the size of thebinding region (pocket or groove) of the target protein for each sidechain. For example, side chains with a ring structure such as benzene,indole and cyclohexane, determine the strength of G2 abrogation or Mabrogation and/or non specific toxicity; see FIGS. 9 and 4, where ringstructures larger than 5 membered affect the G2 abrogating activity(moderate size at P1 and P2 increase G2 abrogating activity, whereas,too large of a structure (P1, P5 and P6) increase M abrogation and/ornon specific toxicity.

Side chains without a ring structure appear neutral. So, to attainbetter activity a proper sized ring structure at P1, P2, P4 and P6, andeither no ring structure at P3 and P5 or a ring structure less than 6members is desired. A proper ring for P1, P2, and P6 is from a one to a6 membered ring through fusion of two rings with either 5 or 6 membered.For P4, a proper size ring is a fusion of two rings, each of which are 5or 6 membered. Thus, for P1, Cha or Nal(2) appear to be the best fits;for P2, Phe-2,3,4,5,6F, Phe-3,4,5F or Phe-CF3 appear best. These sidechain sizes indicate that there are either two pockets or a singlelarger pocket in the target molecule where this region interacts. For P3and P5, a small side chain such as Ser or Pro is acceptable and a largerside chain such as Arg is also acceptable, indicating that there is nopocket in this region of target molecule, so side chains can just layopposite to target. However, it is possible that a ring structure mightenable the peptide or peptidomimetic to interact with another molecule(i.e., other than a target molecule) which may in turn increase sideeffect. For P6, Bpa or Ser-Tyr appear better than Tyr alone or a smallerside chain, indicating a deeper groove that lay horizontally in thetarget. There also may be a shallow and wider pocket for P4 in thetarget based on the sizes of the residues for P4.

The following peptides were analyzed using Jurkat and bleomycin asdescribed. Sequences of peptides are as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser) (d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg) (d-Arg) (d-Arg)(d-Gln) (d-Arg) (d-Arg) (SEQ ID NO:80); CBP700, (d-Arg) (d-Arg)(d-Bpa)(d-Arg) (d-Arg) (d-Arg) (d-Phe-2,3,4,5,6-F) (d-Cha) (SEQ IDNO:96); CBP701, (d-Arg) (d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Trp) (d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:97); CBP702, (d-Arg) (d-Arg)(d-Arg) (d-Arg) (d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:98); and CBP703, (d-Arg) (d-Arg) (d-Arg)(d-Bpa)(d-Arg)(d-Arg) (d-Arg)(d-Phe-2,3,4,5,6-F) (d-Cha) (SEQ ID NO:99). The resultsindicate that CBP700, 701, 702, 703, although shorter than otherexemplified peptides, retain G2 checkpoint abrogating activitycomparable to other peptides having significant G2 checkpoint abrogatingactivity (FIG. 11).

A comparison between G2 checkpoint abrogating activity and non specifictoxicity (M checkpoint abrogation) by CB501 was performed. In brief,Jurkat cells were treated with 40 μg/ml bleomycin or 0.5 μg/mlcolchicine for G2 checkpoint abrogating activity and non specifictoxicity, respectively. The DNA amount in each of the treated cells wasanalyzed by flow cytometry, as previously described. The data indicatethat G2 checkpoint was abrogated in a dose dependent manner for CBP501while non specific toxicity was absent up to 50 μM of peptide, asdetermined by the unchanged percentage of M phase arrested cells (FIG.12).

Example 3

This example describes peptide/peptidomimetic kinase inhibition activityand serum stability analysis of various peptides.

Since two kinases, Chk1 and Chk2, are important for G2 checkpointmechanism, kinase inhibition analysis of both enzymes was performed. Invitro kinase inhibition analysis was performed using “PepTag^((R))Non-Radioactive Protein Kinase Assays”, Promega, according to company'sprotocol, except purified CHK2 kinase was used instead of PKC. PurifiedPKC was purchased from Upstate Biotechnology, Inc. These results areshown in Table 4A.

TABLE 4A Kinase inhibition analysis of the compounds IC₅₀ in μM PKA CHK2CBP450 >400 10 CBP440 180 8

In vitro kinase inhibition analysis was performed by CycLex, Co. Ltd.,Nagano, Japan. Briefly, baculovirus derived recombinant human fulllength Chk1 with histidine tag or E. Coli derived recombinant human fulllength Chk2 fused with GST were used as kinases. E. Coli derivedrecombinant GST-Cdc25C (amino acis 167-267) was used as a substrate.Reaction conditions were 20 mM Hepes-KOH (pH7.5), 1 mM DTT, 80 μg/mlBSA, 10 mM MgCl₂ and 50 mM ATP at 30 degree for 60 min. Thephosphorylation of serine 216 on GST-Cdc25C was detected byanti-Cdc25C-phosphorylated S216 antibody with enzyme linked immuneassay. These results are shown in Table 4B.

TABLE 4B Kinase inhibition analysis of peptides. CHK1 CHK2 CBP500 5.6 8CBP501 7.9 18.6 CBP505 63.4 >100 CBP506 37.6 67 CBP603 15.5 18.1

The data indicate that both Chk1 and Chk2 kinase inhibition occur at adose higher than the G2 abrogating dose (IC₅₀ for G2 abrogation byCBP500, 501, 505, 506, 603 are all less than 1 μM). These resultssuggest that these peptides have a mechanism of action in addition toinhibiting Chk1/2 molecules. Alternatively, the peptides possiblyaccumulate within cells such that their concentration is greater withincells than in the surrounding medium.

Serum analysis was performed to determine the stability of peptides inmouse and human serum. Briefly, peptides (10 mM or 2.5 mM) wereincubated with freshly prepared human serum at 37 degrees for 1 hr.CBP501 (10 mM) was incubated with freshly prepared mouse serum for 1 hrat 37 degree. Jurkat cells were treated with the serum with or withoutpeptides and bleomycin (40 μg/ml) and incubated for 24 hr. Thepopulation of G2 phase cells was determined by flow cytometry aspreviously described. The residual G2 checkpoint abrogating activity ofserum treated peptides were determined by comparing the % G2 cells ofthe treated serum and the standard curve produced with medium treatedpeptides, bleomycin and Jurkat cells (Table 5A). The residual CBP501quantity was determined with HPLC after deproteinating with ethanoltreatment (Table 5B). The data indicate that peptide with d-type aminoacids such as CBP501 and CBP603 are more stable in serum than peptidewith 1-type amino acid such as CBP413.

TABLE 5A Human serum treatment analysis Residual activity of peptideafter 1 hr human serum treatment CBP413 <0.4% of original  CBP501 >50%of original CBP603 >50% of original

TABLE 5B Mouse serum treatment analysis Residual peptide after 1 hrhuman serum treatment CBP501 >90%

Example 4

This example describes the anti-cell proliferative activity of CBP501 oncultured cells. This example also describes data demonstrating in vivoactivity of the peptides/peptidomimetics.

To demonstrate anti-cell proliferative activity of the compounds,cultured MIAPaCa2 human pancreatic carcinoma cells were treated withCBP501 (10 μM), cisplatin (1, 3 or 9 μg/ml) and oxaliplatin (1, 3 or 9μg/ml) alone, and in combination. Briefly, cells were plated at 300cells/well in 6 well plates, incubated overnight, and treated with thecompounds for three hours. The medium was changed and cultured for anadditional 10 days. Cells were subsequently fixed with 70% methanol,stained with 0.1% crystal violet and visualized. The colony formationanalysis results indicated that CBP501 enhanced the cyto-toxic activityof both cisplatin and oxaliplatin against MIAPaCa2 cells.

Similar studies were performed using normal human umbilical endothelialcells (HUVEC). Since normal cells do not form colonies, they were plated3000 cell/well instead of 300 cell/well. The results indicate thatpeptide by itself does not disturb the growth of normal cells nor didthe peptide augment cytotoxic activity of cisplatin and oxaliplatintowards the cell. The peptides therefore do not appear to exhibitsignificant G2 abrogating activity against normal cells subjected tonucleic acid damaging treatment, incontrast to hyperoliferating cellssuch as cancer cells, which are sensitized to nucleic acid damagingtreatment. The results indicate the specificity of the peptide insensitizing proliferating cells but not normal cells against nucleicacid damaging treatment.

TABLE 6 Growth inhibition analysis of MIAPaCa2 using alamar blue. IC5024 hr 48 hr 72 hr cisplatin  16 μM 31 μM 46 μM CBP501   6 μM 10 μM 13 μMCBP501 with 10 uM 0.6 μM  1 μM  6 μM cisplatin

AlamarBlue analysis was performed to analyze the growth inhibitingactivity of CBP501 with and without cisplatin. Briefly, MIAPaCa2 cellswere exposed to 1, 3, 10, 30, 100 μM of cisplatin or 0.22, 0.67, 2, 6,and 18 μM of CBP501 with or without 10 μM cisplatin for three hours in96 well plates at 2500 cell/well in duplicate manner. The medium waschanged and incubated an additional 24, 48 or 72 hour. Followingincubation, 20 μl of alamarBlue 90% reagent was added to each well foranother 6 hours for detection of cell viability by fluorescentintensity. Fluorescent intensity was measured using a Spectrafluor Plusplate reader with exitation 530 nm and emission 590 nm. The IC₅₀ wascalculated (Table 6).

This study indicates that CBP501 alone inhibits cell growth better thancisplatin in molar dose. CBP501 suppressed cell growth at a much lowerdose when combined with 10 μM of cisplatin, which is approximately thedose of cisplatin used for cancer treatment. Furthermore, growthsuppressing activity of CBP501 was longer than cisplatin; the IC₅₀ at 72hour was much better when CBP501 was used than cisplatin.

The in vivo half life of CBP501 was determined by quantifying CBP501 inmouse serum 1, 3 and 6 hr following intra-peritoneal injection of CBP501(40 mg/kg). The residual intact CBP501 quantity was determined with HPLCafter deproteinating mouse serum drawn from injected mice with ethanoltreatment (Table 7).

TABLE 7 in vivo half life of CBP501 Half-life after 40 mg/kgintra-peritoneal injection CBP501 3 hr

To determine tolerance to peptides, groups of ten mice wereintravenously injected once with CBP501 (5, 8 or 10 mg/kg) orintra-peritonealy injected once with CBP501 (50, 80 or 100 mg/kg).Injected mice were observed for a week for their survival (Table 8).

TABLE 8 Maximal tolerated dose in mouse by single injection MTD MTD (iv)(ip) CBP413 14 mg/kg 146.7 mg/kg CBP501 10 mg/kg  98.8 mg/kg

To study in vivo efficacy of the compounds, MIAPaCa2 human pancreaticcarcinoma cells were implanted subcutaneously in scid mice. Thetreatment was initiated when the size of the primary tumor became 0.1cm3 (Day0) or larger, e.g., 7 or 8 mm in diameter. CDDP (3 mg/kg) andCBP 501 (10 or 40 mg/kg) were intra-peritoneally administered alone orin combination. Tumor sizes were measured using calipers three times aweek, and volumes were calculated using the formula: weight(mg)=[width(mm)2×length (mm)]/2. Mean tumor sizes for each treatment group areplotted (n=4) against the days after the start of treatment (FIG. 10).

The results indicate that CBP501 treatment alone suppresses the growthof human pancreatic cancer cell in vivo. The results further indicatethat CBP501 increased the anti-tumor activity of cisplatin.

Example 5

This example includes a description of lung cancer and studies usingCBP501.

Lung cancer is the leading cause of adult cancer deaths in westerncountries. In the USA, 219,440 new cases were diagnosed in 2009 and159,390 deaths occurred due to this disease, accounting for about 29% ofall cancer deaths (see, e.g., American cancer society, Cancer Facts &Figures 2009). Eighty-seven percent (87%) of all new lung cancer casesare non small cell lung cancer (NSCLC) histologies, of which there arethree major types: adenocarcinoma, squamous cell (epidermoid) carcinomaand, large cell carcinoma (American cancer society, Cancer Facts &Figures 2009). Despite improvements in surgical techniques and combinedtherapies, the prognosis for patients diagnosed with NSCLC remains poor.The five-year survival rate is 47% for cases detected in the earlystage, when the disease is still localized, but the majority of NSCLCpatients (68%) (see, e.g., AJCC Cancer Staging Manual. In: Fleming I D,editor. Philadelphia: Lippincott-Raven; 2002) are diagnosed withadvanced disease (stage III) or metastatic disease (stage IV) requiringchemotherapy. The 5-year survival rates are 8.4% for those patients withstage III disease and 1.6% for stage IV, with the majority of patientswith advanced NSCLC, succumbing to disease within 2 years (see, e.g.,American cancer society, Cancer Facts & Figures 2009; AJCC CancerStaging Manual. In: Fleming I D, editor. Philadelphia: Lippincott-Raven;2002.) The introduction of new therapeutics that can produce significantimprovement in patient survival and quality of life is an unmet need.

Patients with advanced stage (IIIb or IV) NSCLC who have a goodperformance status can obtain benefit from chemotherapy (see, e.g.,Souquet, P J., et al., Lancet 342:19-21, 1993; Marino, P., et al., Chest106:861-865, 1994; Marino, P., et al., Cancer 76:593-601, 1995; Helsing,M., et al., Eur J Cancer 34:1036-1044, 1998; Cullen, M H., et al., JClin Oncol 17:3188-3194, 1999; Pfister, D G., et al., J Clin Oncol22:330-353, 2004). Chemotherapy doublets have been shown to improvesurvival when compared with single agents or no chemotherapy (see, e.g.,Bunn, P A., et al., J Clin Oncol 20:23S-33S, 2002). Currentlyrecommended first line chemotherapy regimens in advanced NSCLC includeplatinum compounds (cisplatin [CDDP] or carboplatin) in combination withgemcitabine, vinorelbine, or taxanes (paclitaxel or docetaxel),irinotecan, etoposide, vinblastine, and/or pemetrexed as referenceregimens (Pfister, D G., et al., J Clin Oncol 22:330-353, 2004).

Randomized trials have shown that the various platinum-doubletcombinations are all of similar efficacy although regimens differslightly in terms of toxicity, convenience and cost. Results foundoverall response rates (ORRs) of between 17% and 32%, median survivaltimes of 7 to 10 months, and 1-year survival rates of 30 to 45% (see,e.g., Scagliotti, G., et al., Semin Oncol 32:S5-S8, 2005; Schiller, JH., et al., N Engl J Med 346:92-98, 2002; Scagliotti, G., et al., J ClinOncol 20:4285-4291, 2002; Kelly, K., et al., J Clin Oncol 19:3210-3218,2001; Fossella, F., et al., J Clin Oncol 21:3016-3024, 2003).

Most instances of triplet chemotherapy have so far not resulted infurther increased survival, but instead increased toxicity. A recentstudy of carboplatin+paclitaxel+bevacizumab, however, did show somesurvival benefit (see, e.g., Sandler, A., et al., N Engl J Med355:2542-2550, 2006), suggesting that the addition of a targeted agentwith non-overlapping toxicities may improve doublet chemotherapy. Activeattempts to optimize the benefit of chemotherapy are being pursuedthrough the use of molecular markers predictive of antitumor activity.Genes predictive of chemotherapeutic efficacy in NSCLC are beginning toemerge (see, e.g., Bepler, G., et al., ASCO Educational Book:350-352,2008; Sommers, K., et al., Proc Am Soc Clin Oncol 26 2008). Noteworthyamong these are markers such as ERCC1, BRCA1/2, RRM1 and TS (Table 9).

TABLE 9 Molecular Markers Predictive of Chemotherapeutic Efficacy inNSCLC Marker Expression Sensitivity Resistance ERCC1 ↑ Platinum agents ↓Platinum Agents BRCA1/2 ↓ Platinum Agents Taxane RRM1 ↑ Gemcitabine ↓Gemcitabine TS ↑ Pemetrexed or 5-FU ↓ Pemetrexed or 5-FU

Example 6

This example includes a description of data indicating that certainhuman patient subpopulations respond favorably to combinations ofpeptides and chemotherpauetic (nucleic acid damaging) agents.Unexpectedly, the data show that a sub-group of the patient populationof a clinical study on non-squamous non-small cell lung cancer NSCLC)having less than 10,000 white blood cell (WBC) counts per cubicmillimeter of blood before the treatment with CBP501 received benefit bythe administration of CBP501.

CBP501 is a synthetic dodecapeptide that is comprised entirely ofD-amino acids (FIG. 13). It is an evolved version of TAT-S216A, whichwas optimized for its activity to reduce the accumulation of G2 (4N)cells in response to treatment with DNA-damaging agents, in a DNAcontent flow cytometry-based assay.

Two phase I dose-ranging and pharmacokinetic studies have been conductedto investigate CBP501 in a total of 78 patients: a monotherapy study ofCBP501, administered as a 60-min i.v. infusion on days 1, 8, and 15,repeated every 4 weeks, and a combination therapy study with cisplatinwith administration once every 3 weeks (see, e.g., Shapiro, G I., etal., Clin Cancer Res. May 15; 17(10):3431-42, 2011).

Phase I Single-Agent Study (CBP04-01): This was a first in man,single-agent phase I dose escalation trial, exploring a regimen of threeinjections (days 1-8-15) every 28 days, in a patient population withadvanced solid tumors. A total of 68 cycles were administered, themedian number of cycles per patient was 2 (range 1-8). Two patientsachieved 7 cycles of treatment with stable disease, one with a diagnosisof pancreas cancer and the other with ovarian cancer. The majority ofpatients (87%) discontinued the study due to disease progression. Nopatients discontinued due to toxicity (see, e.g., Shapiro, G I., et al.,Clin Cancer Res. May 15; 17(10):3431-42, 2011).

Phase I study of CBP501 in combination with cisplatin (CBP06-01): Themain goal of this phase I study was to determine the MTD and RD ofCBP501 and cisplatin when administered in combination once every 21days. CBP501 was administered first, as a 1-hour infusion, followed bycisplatin two hours after treatment start. Patients were also givenprophylactic treatment for allergic reactions according to the sameregimen developed for the phase I single-agent study (loratadine,dexamethasone, ranitidine and diphenhydramine).

A total of 48 patients were treated in three US centers and a total of182 cycles were administered, the median number of cycles per patientwas 4 (range 1-13). CBP501 was explored in a range of doses from 3.6mg/m² to 36.4 mg/m². The highest dose level studied was CBP501 36.4mg/m² and cisplatin 75 mg/m². At this dose level, two out of sixpatients experienced allergic reactions judged by the investigators asdose limiting (grade 3). The MTD was considered as the dose levelimmediately below, which was CBP501 24.3 mg/m² and cisplatin 75 mg/m².Hints of activity were documented in several patients (see, e.g.,Shapiro, G I., et al., Clin Cancer Res. May 15; 17(10):3431-42, 2011).

Cisplatin (cis-diamminodichloroplatinum), an inorganic platinumcoordination complex, reacts preferentially at the N7 position ofguanine and adenine residues of DNA to form a variety of monofunctionaland bifunctional adducts. These adducts contribute to the drug'scytotoxicity, by impeding various cellular processes that require theseparation of both DNA strands such as replication and transcription.

Cisplatin has been assessed clinically against a variety of tumorsbecause of its solid antineoplastic activity against testicular andovarian cancers. Since its approval, cisplatin has been a criticalchemotherapeutic agent and has been widely used, either alone or incombination with other antineoplastic agents. Cisplatin is also known toconfer a substantial palliative effect in patients presenting with othertumor types, e.g. lung cancer, bladder carcinoma and head and neckcarcinoma, and it is included in most chemotherapy regimens used inthese diseases.

Pemetrexed disodium is a structurally novel antifolate possessing aunique 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus, and which inhibitsthe function of folate-dependent enzymes involved in the synthesis ofsubstrates necessary for cell growth and division such as thymidylatesynthase, dihydrofolate reductase, and glycinamide ribonucleotideformyltransferase (see, e.g., Taylor, E C., et al., J Med Chem35:4450-4454, 1992; Schultz, R M., et al., Anticancer Res 19:437-443,1999).

Pemetrexed has demonstrated activity in clinical trials in a largevariety of tumor types, including lung, breast, colon, pleura, pancreas,stomach, bladder, head and neck, and cervix. Pemetrexed in combinationwith cisplatin was approved by the FDA on Feb. 4, 2004 for the treatmentof patients with MPM whose disease is either unresectable or who areotherwise not candidates for curative surgery.

In phase II studies in chemotherapy-naïve patients with NSCLC,pemetrexed in combination with cisplatin or carboplatin has yieldedefficacy results comparable with other platinum doublets (see, e.g.,Scagliotti, G., et al., Clin Cancer Res 11:690-696, 2005; Zinner R., etal., Cancer 104:2449-2456, 2005; Mangold, C., et al., Ann Oncol11:435-440, 2000; Shepherd, F A., et al., Cancer 92:595-600, 2001). Inaddition, pemetrexed has an excellent safety profile and a convenientadministration schedule.

A recent randomized phase III study compared, in a non inferioritydesign trial, the overall survival (OS) between 1725 chemotherapy-naïvepatients with stage III or IV NSCLC treated with cisplatin plusgemcitabine or cisplatin plus pemetrexed every 3 weeks for up to sixcycles (see, e.g., Scagliotti, G., et al., J Clin Oncol 26:3543-3551,2008; Pimentel, F., et al., Proc Am Soc Clin Oncol 26 (Part I ofII):448s, 2008, (Suppl. 15S)(abstr) #448s). The OS for cisplatin pluspemetrexed was not inferior to cisplatin plus gemcitabine (mediansurvival, 10.3 months for both treatments). OS was statisticallysuperior for cisplatin plus pemetrexed versus cisplatin/gemcitabine inpatients with adenocarcinoma (n=847; 12.6 months and 10.9 months,respectively) and large cell carcinoma histology (n=153; 10.4 months and6.7 months, respectively). For cisplatin plus pemetrexed, rates of grade3 or 4 neutropenia, anemia, and thrombocytopenia; febrile neutropenia;and alopecia were significantly lower than for the cisplatin/gemcitabinetreatment arm, whereas grade 3 or 4 nausea was more common.

Patients and Methods:

Clinical Study Design: Open-label, multicenter, phase II randomized,two-arm, comparative study. The protocol evaluated full-dose cisplatinand pemetrexed with or without CBP501. Patients were randomized in a 1:1ratio to pemetrexed, cisplatin and CBP501 (Arm A) or pemetrexed andcisplatin (Arm B). Randomization was stratified according to baselinestage of disease (IIIb vs IV), presence of brain metastasis and whetheror not patients were eligible for bevacizumab therapy.

Investigator/Trial Location: Approximately 40 centers in the USA,Russia, Canada, Brazil, Argentina and Peru.

Study Objectives:

Primary: To compare the efficacy, progression free survival, ofcisplatin and pemetrexed with or without CBP501 in patients with locallyadvanced (stage IIIB with malignant pleural effusion or pericardialeffusion) or metastatic (stage IV) non-squamous NSCLC.

Secondary:

To characterize the safety profile of the study regimen and the efficacyparameters other than progression free survival such as overallsurvival.

Study Population:

Inclusion Criteria:

1. Signed informed consent obtained prior to initiation of anystudy-specific procedures

2. Histologically or cytologically confirmed diagnosis of non-squamousnon small cell lung cancer (NSCLC), not amenable for radical resection,stage IIIB with pleural or pericardial effusion or stage IV, who has notreceived previous chemotherapy or other systemic treatment

3. At least one unidimensionally measurable lesion according to theResponse Evaluation Criteria in Solid Tumors (RECIST)

4. Male or female patients aged at least 18 years

5. ECOG Performance Status (PS): 0-1

6. Life expectancy>3 months

7. Prior local radiotherapy is allowed if it was completed ≧3 weeksprior to the first dose of the study medication

8. Concomitant palliative radiotherapy to an existing bone lesion forpain control is allowed

9. Prior surgery is allowed if it is performed at least 4 weeks prior tothe first dose of study medication and patient should be fully recovered

10. Adequate organ function, including the following:

-   -   Bone marrow: white blood cell (WBC) count ≧4×109/L, absolute        neutrophil count (ANC) ≧1.5×109/L, platelet count ≧100×109/L,        hemoglobin ≧9 g/dL    -   Hepatic: Bilirubin ≦1.5×the upper limit of normal (ULN),        aspartate transaminases (AST/SGOT) and alanine transaminases        (ALT/SGPT)≦2.5×ULN (or ≦5×ULN if liver metastases are present),        INR≦1.5×ULN, albumin ≦3.0 g/dL    -   Renal: Serum creatinine ≦1.5 mg/dL or creatinine clearance ≧45        mL/min (calculated according to the Cockroft and Gault formula)

11. Female patients of child-bearing potential must have a negativepregnancy test and be using at least one form of contraception asapproved by the Investigator for 4 weeks prior to the study and 4 monthsafter the last dose of study drug. For the purposes of this study,child-bearing potential is defined as: “All female patients unless theyare post-menopausal for at least one year or are surgically sterile”

12. Male patients must use a form of barrier contraception approved bythe Investigator during the study and for 4 months after the last doseof study drug

13. Ability to cooperate with the treatment and follow-up

Exclusion Criteria:

1. Radiation therapy to more than 30% of the bone marrow prior to entryinto the study

2. Presence of neuroendocrine features in the tumor sample

3. Previous treatment with chemotherapy, new biological therapies (smallmolecules, antibodies), immunotherapy

4. Absence of measurable lesions

5. An ongoing or active infection, symptomatic congestive heart failure,unstable angina pectoris, symptomatic or poorly controlled cardiacarrhythmia, uncontrolled thrombotic or hemorrhagic disorder, or anyother serious uncontrolled medical disorders in the opinion of theInvestigator

6. Any previous history of another malignancy within 5 years of studyentry (other than cured basal cell carcinoma of the skin or curedin-situ carcinoma of the cervix)

7. Presence of any significant central nervous system (CNS) orpsychiatric disorder(s) that would hamper the patient's compliance

8. Evidence of peripheral neuropathy>grade 1 according to NCI-CTCAEVersion 3

9. Treatment with any other investigational agent, or participation inanother clinical trial within 28 days prior to study entry

10. Pregnant or breast-feeding patients or any patient with childbearingpotential not using adequate contraception

11. Known HIV, HBV, HCV infection

12. Presence of symptomatic brain metastasis. Patients with brainmetastases must:

-   -   Have stable neurologic status following local therapy (surgery        or radiation) for at least 2 weeks after completion of the        definitive therapy and have discontinued use of corticosteroids        for 1 week prior to the study entry.    -   Be without neurologic dysfunction that would confound the        evaluation of neurologic and other AEs

13. Inability or unwillingness to take folic acid, vitamin B12 orcorticosteroids

14. Inability to interrupt aspirin or other nonsteroidalanti-inflammatory agents, other than aspirin dose ≦1.3 grams per day,for a 5-day period (8-day period for long-acting agents, such piroxicam)

15. Significant weight loss (≧10% body weight during preceding 6 weeks)

16. Presence of clinically significant (by physical exam) third spacefluid collections, e.g., ascites or pleural effusions that cannot becontrolled by drainage or other procedures prior to study entry

Number of Patients:

A total of 195 patients were treated in which 97 patients were treatedwith CBP501, cisplatin and pemetrexed (Arm A) and 98 patients weretreated with pemetrexed and cisplatin (Arm B).

Study Drug:

Formulation: CBP501 for injection was provided in single dose vials (20mg) containing a sterile lyophilized powder comprising CBP501 peptideacetate salt (peptide base units). For administration, vial contentswere reconstituted in 5% Dextrose Injection, USP, and added to a 100 mLi.v. bag of 5% Dextrose Injection, USP.

Pemetrexed: A commercial formulation of pemetrexed was used, withreconstitution in 20 mL 0.9% sodium chloride solution for injection,then dilution to 100 mL.

Cisplatin: A commercial formulation of cisplatin was used and wasdiluted in 250 mL of normal saline for administration.

Dose Regimen and Route of Administration:

CBP501, pemetrexed and cisplatin was administered on the same day (Day1), every 3 weeks for a maximum of six cycles. A cycle was considered tobe 3 weeks (21 days).

Arm A

1. CBP501 25 mg/m² was administered as an i.v. infusion of 1 hour.

2. Pemetrexed 500 mg/m² was administered as an i.v. infusion over 10minutes, immediately after the CBP501 infusion.

3. Cisplatin 75 mg/m² was administered as a 1-hour i.v. infusionimmediately after the pemetrexed infusion.

Arm B

1. Pemetrexed 500 mg/m² was administered as an i.v. infusion over 10minutes.

2. Cisplatin 75 mg/m² was administered as a 1-hour i.v. infusionimmediately after the pemetrexed infusion.

Each combination was administered via a central or peripheral venousaccess.

Prophylactic Treatment:

All Patients Enrolled Received:

1. Vitamin supplementation: all patients were instructed to take alow-dose oral folic acid preparation or multivitamin with folic acid ona daily basis. At least 5 daily doses of folic acid must have been takenduring the 7-day period preceding the first dose of pemetrexed, anddosing should continue during the full course of therapy and for 21 daysafter the last dose of pemetrexed. The suggested dose of folic acid wasin the range 350-1000 μg. Patients must have also received one (1)intramuscular injection of vitamin B12 during the week preceding thefirst dose of pemetrexed and every 3 cycles thereafter. Subsequentvitamin B12 injections may have been given the same day as pemetrexed.The dose of vitamin B12 was 1000 μg.

2. Dexamethasone 4 mg orally, twice per day, the day before, the day oftreatment administration and the day after.

3. Prophylactic antiemetic treatment: consisting of 5HT3antagonists+steroids according to standard treatment center protocols.Patients were given further oral antiemetics as needed.

-   -   The following hydration protocol was suggested in patients        without cardiovascular impairment. Similar protocols routinely        administered in the investigator centers could have been        implemented:

1. Patients received a total of 1.5-2.0 liters hydration (5% dextrose or½ normal saline) with 20 mEq KCl/liter and 1 g MgSO4/liter, ran at 500mL/hour.

2. After the patient had received 1-hour of the hydration infusion, 12.5g of mannitol was administered by IV push.

3. The cisplatin infusion (mixed in normal saline at 1 mg/mL) was theninfused over 1 hour, while continuing the hydration infusion.

4. Additional mannitol was administered (12.5-50.0 g by IV push), ifnecessary to maintain urinary output at 250 mL/hour over the duration ofthe hydration.

-   -   For patients treated with CBP501 (Arm A), it was recommended        that they receive the following prophylactic regimen to reduce        the incidence and severity of symptoms due to histamine release:

1. Diphenhydramine (DPH) 50 mg IV and Ranitidine 50 mg IV (or anotherhistamine H2 antagonist) before each CBP501 infusion.

2. Loratadine (10 mg) PO the day before (day −1), the day of CBP501administration (day 0) and the day after (day 1).

Duration of Study Period Per Patient:

Patients will receive a maximum of six cycles of study treatment unlessany of the following are observed earlier:

-   -   disease progression    -   unacceptable toxicity    -   withdrawal of consent    -   serious protocol violation    -   treatment delay>2 weeks (except in the case of potential or        perceived patient benefit)

Following treatment discontinuation, patients will be followed every 8weeks until disease progression or initiation of further systemicanticancer therapy, and then every 6 months until death.

Informed Consent

The Investigator thoroughly explained to the patient the purpose andmethods of the study, as well as any expected effects and adversereactions, before any study specific screening procedures wereconducted. The patient was provided with an information sheet and wasgiven sufficient time and opportunity to inquire about the details ofthe trial and to decide whether or not to participate. The patient andthe person with whom they discuss the informed consent signed and datedthe consent form.

The Investigator explained that the patient was completely free torefuse to enter the study or to withdraw from it at any time and for anyreason. Similarly, the Investigator and/or Sponsor were free to withdrawthe patient at any time for safety or administrative reasons. Any otherrequirements necessary for the protection of the human rights of thepatient was explained, according to current CFR (21, parts 312D, 50 and56) and ICH (ICH E6 1997) GCP guidelines and the Declaration ofHelsinki, 1964 (as clarified in Tokyo in 2004).

Assignment of Patient Numbers

Patient randomization and assignment to a treatment arm were centrallymanaged.

Statistical Analysis:

Cox proportional hazards model was employed to estimate the hazard ratio(HR) for PFS between the 2 treatment arms. The model included thetreatment arm as a factor as well as the randomization stratificationfactors. The following covariates were explored: age, gender, race(Caucasian/non-Caucasian), prior surgery/procedure (yes/no), priorradiotherapy (yes/no), x-ray interpretation (normal/abnormal), ECGinterpretation (normal/abnormal), bone scan (normal/abnormal), and timefrom diagnosis. Any continuous variables such as age and time fromdiagnosis to study treatment could have been converted into categoricalvariables by specifying 2 or several classes of values if a better modelfit would result. The exploratory variables were entered using astepwise regression algorithm using the following criteria: a variablemust be significant at the 0.25 level to be entered into the model andsignificant at 0.15 level to remain in the model. For the final model,the point estimate of the hazard ratios were provided along with 95%CIs.

Subgroup analyses were conducted for patients that had WBC<10000 μL atscreening. Additional subgroup analysis was performed with a softwareGraphPad Prism 5 with raw data on each patients analyzed.

Efficacy Results:

The Cox proportional hazards model analysis without exploring othercovariates on progression free survival (PFS) indicated that Arm A (thearm with CBP501) had a higher hazard than Arm B (HR=1.20 [0.88, 1.65]),but it was not statistically significant (P=0.25). The same modelexploring other covariates also indicated that Arm A had a higher hazardthan Arm B (HR=1.21 [0.85, 1.73]), but it was not statisticallysignificant (P=0.30).

For patients that had WBC<10000/μL at screening, the Cox proportionalhazards model analysis without exploring other covariates indicated thatArm A had a higher hazard than Arm B (HR=1.04 [0.73, 1.49]), but it wasnot statistically significant (P=0.81). The same model exploring othercovariates also indicated that Arm A had a higher hazard than Arm B(HR=1.06 [0.71, 1.59]), but it was not statistically significant(P=0.78).

It was noted that the hazard ratio for PFS improves for Arm A when theanalysis was restricted to patients who had WBC<10000/μL at screening inboth of the analysis (Tables A, B).

TABLE A Cox covariates analysis on PFS without exploring othercovariates PFS with independent radiological review Cox covariatesanalysis without exploring other covariates Hazard Ratio P-value ALLtreated 1.20 0.25 WBC <10000 1.04 0.81

TABLE B Cox covariates analysis on PFS exploring other covariatesExploring other covariates Hazard Ratio P-value ALL treated 1.21 0.30WBC <10000 1.06 0.78

Cox Proportional Hazards Model Analysis on Overall Survival (OS) on allTreated Population

Arm A had a lower hazard than Arm B without and with exploring othercovariates (HR=0.96 and 0.77). The difference was not statisticallysignificant (P=0.82 and 0.25).

Cox Proportional Hazards Model Analysis on OS on Patients that HadWBC<10000/μL at Screening

For OS on patients that had WBC<10000/μL at screening in the TreatedPopulation, Arm A had a lower hazard than Ann B without and withexploring other covariates (HR=0.80 and 0.69); the difference was notstatistically significant (P=0.32 and 0.16).

It was noted that the hazard ratio for OS improves for Arm A when theanalysis was restricted to patients who had WBC<10000/μL at screening inall of the analysis (Tables C, D).

TABLE C Cox covariates analysis on OS without exploring other covariatesCox covariates analysis on overall survival without exploring othercovariates Hazard Ratio P-value ALL treated 0.96 0.82 WBC <10000 0.800.32

TABLE D Cox covariates analysis on OS exploring other covariatesExploring other covariates Hazard Ratio P-value ALL treated 0.77 0.25WBC <10000 0.69 0.16

FIG. 14 shows Kaplan-Meyer survival curves, median OS and hazard ratioin relation to the WBC at screening (baseline) in all treated patients.The hazard ratio improves as the cut off level decreases and peaks atWBC 8000/μl as cut off level.

Referring to FIG. 17, Neutrophils were purified with EasySep neutrophilenrichment kit (Stemcell technol.) from human peripheral blood afterremoval of red blood cells with Hetasep (Stemcell technol.). Purifiedneutrophils (1×10⁶ cell/well, 24 well plates were cultured with orwithout 1 μM of CBP501 for 15 minutes (terminated reaction by addingEDTA) and further four hours with 1 nM PMA, 3 or 10 μM A23187, or 100 or1000 ng/ml LPS. The wells were washed two times and incubated with DNasefor 15 min and the supernatants were collected, incubated with Elastasesubstrate for 2 hours, and then analyzed to detect elastase activity.

Referring to FIG. 18, C57BL/6 mice, 8 weeks of age, were intravenouslyinjected with or without LPS 2.5, 5, or 10 ug/ml 30 minutes before theinjection of diphenhydramine. CBP501 (7.5 mg/kg) was injected 30 minutesafter diphenhydramine injection. The thrombin/antithrombin complex wasquantified with ELISA in the plasma that was separated from the blooddrawn 3 hours after the CBP501 or mock injection. The data were obtainedin each condition out of four animals.

Referring to FIG. 19, macrophages were obtained by stimulating humanperipheral blood mononuclear cells by 0.32 uM of PMA and removing allsuspension cells 48 hours of PMA stimulation. The cells were furtherincubated with 50 ng/ml IFN-gamma, 10 ng/ml LPS to obtain M1 phenotype,and 20 ng/ml IL-4 to obtain M2 macrophages. Both treatment was with orwithout CBP501. The phagocytic activity was monitored by usingfluorescent labelled beads and flow cytometry.

Referring to FIG. 20, a macrophage cell line RAW264.7 was incubated withor without 0.1 or 1 μM of CBP501 for 3-6 hours, and then furtherincubated with or without 10 or 1000 ng/ml LPS for 4 hours. The releasedTNF were measured by ELISA.

CBP501 showed potential as an effective anti-tumor agent in preclinical(Sha, S., et al. Mol. Cancer Ther. 6:147 (2007)) and Phase I clinicalstudies (Shapiro, G. I., et al. Clin. Cancer Res. (2011)). CBP501 mayoperate under two mechanisms of action, e.g. via cell cycle G2checkpoint abrogation (Sha, S., et al. Mol. Cancer Ther. 6:147 (2007))and platinum concentration in tumor cells or through Calmodulininhibition (Mine, N., et al. Mol. Cancer Ther. 10:1929 (2011)).

As demonstrated herein, it was unexpectedly discovered by way ofsub-group analysis on the patient population of a Phase II clinicalstudy on non-squamous non-small cell lung cancer patients that there wasa statistically significant (p<0.0001) difference in the survival ofgroups of patients between that with high white blood cell count (WBC)at screening and that with normal or low WBC.

Further as indicated by the results herein, CBP501, in addition todirect action on tumor cells, may also act on the tumormicro-environment, such as macrophages, by inhibiting calmodulin andincreasing the neutrophil extracellular traps (NETs) when patients areinflamed by reducing clearance/phagocytosis of NETs by macrophages. Thismay increase the chance of having deep vein thrombosis (DVT) andmetastasis, and thus potentially affect patient survival adversely. Onthe contrary, inhibition of M2 type macrophage in patients may preventthe positive action of macrophages on the tumor growth, angiogenesis,metastasis and tumor immune evasion, all of those promotes tumormetastasis which may shorten survival of patients.

Consistent with this observation, increased NETs formation of activatedneutrophils by CBP501 treatment in vitro and increasedthrombin/Anti-thrombin complex formation in LPS stimulated mice havebeen demonstrated Inhibition of cytokine secretion and phagocytosis ofboth M1 and M2 types of macrophages by CBP501 has also been indicated.

While clinical studies have indicated CBP501 operating by enhancingcisplatin's cytotoxicity against tumor cells, results herein demonstratean unexpected finding by a sub-group analysis done on the Phase II studyon non-squamous NSCLC patients indicating that groups of patients withhigh white blood cell counts (WBC) at screening of the clinical studysurvived shorter and the other groups of patients with normal or low WBCsurvived longer in response to the regimen with CBP501, and thedifference was statistically highly significant with a p valuecalculated on a Kaplan-Meyer's curves on the overall survival byLog-rank (Mantel-Cox) test was less than 0.0001.

It was thus unexpectedly found that patients with normal or low WBCbefore treatment benefited from CBP501 treatment, while the patientswith high WBC could have been adversely affected by the same treatment.CBP501's inhibitory activity on Calmodulin suggests that the effect on avariety of micro-environmental cells, such as macrophages, leukocytesand lymphocytes, may have inhibited or modulated their activity whichcould have prompted the bidirectional results because, for example, byjust inhibiting macrophages, if it inhibited M1 type macrophages it mayhave adversely affected patient survival and if it inhibited M2macrophages it would prolong patient survival.

Calmodulin inhibitors have been reported to inhibit multiple functionsof macrophages (Horwitz, S. B., et al. J. Cell Biol. 91:798 (1981);Takenawa, T., et al. Biochem J. 15:208 (1982); Westra, J., et al. BMCMusculoskelet. Disord. 30:11 (2010)), leukocytes (Naccache, P. H., etal. Biochem. Biophys. Res. Commun 97(1):62 (1980); Takeshige, K., et al.Biochem. Biophys. Res. Commun. 99(2):484 (1981); Jones, H. P., et al.Biochem Biophys. Acta. 714(1):152 (1982); Jones, H. P., et al. MethodsEnzymol. 015:389 (1984); Verploegen, S., et al. Eur. J. Biochem. 269(18)4625 (2002)) and lymphocytes (Salisbury, J. L., et al. Nature 12:294(1981); Boubali, S., et al. Mol. Immunol. 52(2):51 (2012)). The patientswith high WBC will tend to have M1 macrophages as they arepro-inflammatory type and the patients with normal or low WBC with tumorwill tend to have M2 macrophages (Hao, N., et al. Clin. Dev. Immunol.(2012)). Also, patients with high WBC are known to be more prone to havedeep vein thrombosis (DVT) (Pabinger, I., et al. Blood 122:12 (2013);Blix, K., et al. PLOS One 4:8 (2013); Wang, T. F., et al. Thromb. Res.133(1):25 (2014)), which is a cause of death for significant number ofcancer patients. Those patients tend to have more NETs, and NETs promotetumor metastasis (Cools-Lartigue, J., et al. J. Clin. Invest. (2013))which is one reason for short prognosis of many cancer patientsincluding those with lung cancer.

In this Example there was no statistically significant survival benefitdetected from the addition of CBP501 to the standard regimen, pemetrexedplus cisplatin, when it was analyzed in all treated population. However,it was unexpectedly identified by sub-group analysis that the additionof CBP501 provided a benefit to a group of people who showed normal orlow counts of white blood cell (WBC) at the screening for the clinicaltrial. The normal value of the WBC varies by sites and countries. Theupper normal WBC limits could be from 8000/μl to 11000/μl.

It was surprising that patients with normal range of WBC benefited fromCBP501, and those with high WBC at screening performed worse than thepatients treated with cisplatin and pemetrexed, although both of thedifferences were not statistically significant when compared to controlarm, cisplatin and pemetrexed treated population.

While the precise reason of this potentially bidirectional action ofCBP501 is not evident, CBP501's inhibitory action of Calmodulinindicates that the inhibition of Calmodulin in a variety ofmicroenviromental cells, such as macrophages, leukocytes andlymphocytes, inhibits or modulates their activity which prompted thebidirectional result because, for example, if it inhibited M1 typemacrophages it would adversely affected patient survival by inhibitinganti-tumor activity of macrophages and/or inhibiting clearance of NET,as NET promotes thrombo-genesis and metastasis, and if it inhibitedpro-tumor M2 macrophages it would prolong patient survival. In addition,cisplatin is known to skew macrophages from M1 to M2 type (Dijkgraaf, E.M., et al. Cancer Res. 15:73(8):2480 (2013)), and chemotherapy by itselfis known to promote tumor metastasis (Haas, M. J. SciBX 1-3 (2011)),thus the presence of CBP501 while chemotherapy is on might havesignificant impact on the tumor metastasis. Calmodulin inhibitors areknown to be able to inhibit multiple functions of macrophages (Horwitz,S. B., et al. J. Cell Biol. 91:798 (1981); Takenawa, T., et al. BiochemJ. 15:208 (1982); Westra, J., et al. BMC Musculoskelet. Disord. 30:11(2010)), leukocytes (Naccache, P. H., et al. Biochem. Biophys. Res.Commun 97(1):62 (1980); Takeshige, K., et al. Biochem. Biophys. Res.Commun. 99(2):484 (1981); Jones, H. P., et al. Biochem Biophys. Acta.714(1):152 (1982); Jones, H. P., et al. Methods Enzymol. 015:389 (1984);Verploegen, S., et al. Eur. J. Biochem. 269(18) 4625 (2002)) andlymphocytes (Salisbury, J. L., et al. Nature 12:294 (1981); Boubali, S.,et al. Mol. Immunol. 52(2):51 (2012)). The patients with high WBC willtend to have more M1 macrophages as they are pro-inflammatory and thepatients with normal or low WBC with tumor will tend to have M2macrophases (Hao, N., et al. Clin. Dev. Immunol. (2012)). Also, patientswith high WBC would tend to have more NETs. If the phagocytosis of NETswas prevented by CBP501, patients would be more on the risk of havingDVT and metastasis both of which would reduce survival time.

Alternatively, as calmodulin is involved in normal functions of whiteblood cells (Horwitz, S. B., et al. J. Cell Biol. 91:798 (1981);Takenawa, T., et al. Biochem J. 15:208 (1982); Westra, J., et al. BMCMusculoskelet. Disord. 30:11 (2010); Naccache, P. H., et al. Biochem.Biophys. Res. Commun. 97(1):62 (1980); Takeshige, K., et al. Biochem.Biophys. Res. Commun 99(2):484 (1981); Jones, H. P., et al. BiochemBiophys. Acta. 714(1):152 (1982); Jones, H. P., et al. Methods Enzymol.015:389 (1984); Verploegen, S., et al. Eur. J. Biochem. 269(18) 4625(2002); Salisbury, J. L., et al. Nature 12:294 (1981); Boubali, S., etal. Mol. Immunol. 52(2):51 (2012); Hao, N., et al. Clin. Dev. Immunol.(2012)). CBP501 may interfere and set off its beneficial activity onoverall survival by the potential harm to the function of WBC when theywere excessively required, e.g. the situation when patients weresuffered from active infection which will increase white blood cellcount.

Calmodulin inhibition may also affect anti-cancer immunity by acting onlymphocytes, as it has been suggested that Calmodulin may induce T cellanergy (Boubali, S., et al. Mol. Immunol. 52(2):51 (2012)).

Pre-treatment or base line WBC count has been indicated to be aprognosis factor for patients with NSCLC treated with platinum basedtherapy (Teramukai, S., et al. Eur. J. Cancer (45(11:1950 (2009); Kim,J. W., et al. Cancer Res. Trest. 45:4):325 (2013)) CBP501 may enhancethis effect by modulating cisplatin's activity. Since every patient willget the laboratory analysis of WBC counts as a universally approvedstandard procedure before the treatments, patients may be selected basedon WBC counts.

Calmodulin inhibition by CBP501 may also directly inhibit tumormigration and metastasis, independent of the platinum concentration, ascalmodulin has been shown to play important role in migration (Wang, H.,et al. Nat. Commun. 4:1354 (2013)).

What is claimed is:
 1. A method for increasing nucleic acid damage of ahyperproliferating cell or for the prophylaxis or treatment of a cellproliferative disorder in a mammal, comprising administering a peptidecompound, wherein the peptide compound comprises (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:1); andwherein the mammal has a white blood cell count of less than about11,000 white blood cells per microliter (wbc/μl) of blood.
 2. The methodof claim 1, wherein the mammal has a white blood cell count betweenabout 4,000 to about 11,000 white blood cells per microliter (wbc/μl) ofblood.
 3. The method of claim 1, wherein the mammal has a white bloodcell count of less than about 10,000 white blood cells per microliter(wbc/μl) of blood.
 4. The method of claim 1, wherein the mammal has awhite blood cell count of less than about 9,000 white blood cells permicroliter (wbc/μl) of blood.
 5. The method of claim 1, wherein themammal has a white blood cell count between about 4,000 to about 9,000white blood cells per microliter (wbc/μl) of blood.
 6. The method ofclaim 1, wherein the mammal has a white blood cell count of less thanabout 8,000 white blood cells per microliter (wbc/μl) of blood.
 7. Themethod of claim 1, wherein the mammal has a white blood cell count ofless than about 7,000 white blood cells per microliter (wbc/μl) ofblood.
 8. The method of claim 1, wherein the peptide compound comprisesa pharmaceutical formulation.
 9. The method of claim 1, wherein thepharmaceutically acceptable salt thereof is selected from acetate,sulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogen-phosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, propionate, decanoate,caprylate, acrylate, formate, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates,phthalates, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, -hydroxybutyrate, glycolate, tartrate,methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, and mandelate.
 10. The method of claim 1,wherein the peptide compound has a length from 6 to 10, 10 to 15, 15 to20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to150, 150 to 200, or 200 to 300 amino acid residues.
 11. The method ofclaim 1, wherein the peptide compound further comprises a cellpenetrating molecule attached or conjugated thereto.
 12. The method ofclaim 11, wherein the cell penetrating molecule is joined to the peptidecompound by a covalent bond, or a peptide or a non-peptide linker. 13.The method of claim 11, wherein the cell penetrating peptide comprisesan alternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.
 14. The method of claim 11, wherein the cellpenetrating peptide comprises a polycationic or amphipathic alpha-helixstructure.
 15. The method of claim 1 or 11, wherein the cell penetratingpeptide comprises L- or D-isomer amino acids, or a mixture of L- andD-isomer amino acids.
 16. The method of claim 11, wherein the cellpenetrating peptide comprises a poly-Arginine (Arg) sequence.
 17. Themethod of claim 11, wherein the cell penetrating peptide comprises orconsists of (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg).
 18. The methodof claim 1, further comprising administering a nucleic acid damagingagent, a nucleic acid damaging treatment, an anti-proliferative agent,or an anti-proliferative treatment.
 19. The method of claim 18, whereinthe nucleic acid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or anti-proliferative treatment comprisessurgical resection, radiotherapy, ionizing or chemical radiationtherapy, chemotherapy, immunotherapy, local or regional thermal(hyperthermia) therapy, vaccination, an alkylating agent, ananti-metabolite, a plant extract, a plant alkaloid, nitrosourea, ahormone, or a nucleoside or nucleotide analogue.
 20. The method of claim1, wherein the peptide compound is administered prior to, with or aftera nucleic acid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or an anti-proliferative treatment isadministered.
 21. The method of claim 1, wherein the peptide compound isadministered less than 48 hours prior to or after a nucleic aciddamaging agent, a nucleic acid damaging treatment, an anti-proliferativeagent, or an anti-proliferative treatment is administered.
 22. Themethod of claim 1, wherein the peptide compound is administered lessthan 24 hours prior to or after a nucleic acid damaging agent, a nucleicacid damaging treatment, an anti-proliferative agent, or ananti-proliferative treatment is administered.
 23. The method of claim 1,wherein the peptide compound is administered less than 12 hours prior toor after a nucleic acid damaging agent, a nucleic acid damagingtreatment, an anti-proliferative agent, or an anti-proliferativetreatment is administered.
 24. The method of claim 1, wherein thepeptide compound is administered less than 6 hours prior to or after anucleic acid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or an anti-proliferative treatment isadministered.
 25. The method of claim 1, wherein the peptide compound isadministered less than 4 hours prior to or after a nucleic acid damagingagent, a nucleic acid damaging treatment, an anti-proliferative agent,or an anti-proliferative treatment is administered.
 26. The method ofclaim 1, wherein the peptide compound is administered less than 2 hoursprior to or after a nucleic acid damaging agent, a nucleic acid damagingtreatment, an anti-proliferative agent, or an anti-proliferativetreatment is administered.
 27. The method of claim 1, wherein thepeptide compound is administered less than 1 hour prior to or after anucleic acid damaging agent, a nucleic acid damaging treatment, ananti-proliferative agent, or an anti-proliferative treatment isadministered.
 28. The method of any one of claims 18 to 27, wherein thenucleic acid damaging agent or anti-proliferative agent comprises adrug.
 29. The method of any one of claims 18 to 27, wherein the nucleicacid damaging agent or anti-proliferative agent comprises a platinumcontaining drug.
 30. The method of any one of claims 18 to 27, whereinthe nucleic acid damaging agent or anti-proliferative agent comprisescis-platin, carboplatin, nedaplatin, mitaplatin, satraplatin,picoplatin, triplatin, miriplatin, or oxaliplatin.
 31. The method ofclaim 1, further comprising administering a platinum containing drug,cis-platin, carboplatin, oxaliplatin, pemetrexed, gemcitabine,5-fiuorouracil (5-FU), rebeccamycin, adriamycin (ADR), bleomycin (Bleo),pepleomycin, cisplatin, cisplatinum, or cis-diamminedichloroplatinum(II)(CDDP), oxaliplatin, or camptotecin (CPT), cyclophosphamide,azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil,mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine,5-fluorouracil, cytosine arabinoside, AZT, 5-azacytidine (5-AZC) or a5-azacytidine related compound, actinomycin D, mithramycin, mitomycin C,carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, mitotane, procarbazine, dacarbazine, a taxane, vinblastine,vincristine, doxorubicin, dibromomannitol, radiation or a radioisotope.32. The method of claim 31, wherein the radiation comprises UVradiation,IR radiation, Xray, or alpha-, beta- or gamma-radiation.
 33. The methodof claim 31, wherein the radioisotope comprises I¹³¹, I^(125,)Sr⁸⁹,Sm¹⁵³, Y⁹⁰, or Lu¹⁷⁷.
 34. The method of claim 1, wherein the cellproliferative disorder comprises a tumor or cancer.
 35. The method ofclaim 34, wherein the cell proliferative disorder comprises a metastatictumor or cancer.
 36. The method of claim 34 or 35, wherein the tumor orcancer comprises a lung tumor or cancer.
 37. The method of claim 34 or35, wherein the lung tumor or cancer comprises a small cell or non-smallcell lung cancer.
 38. The method of claim 34 or 35, wherein the lungtumor or cancer comprises an adenocarcinoma, squamous cell carcinoma ora large cell carcinoma.
 39. The method of claim 34 or 35, wherein thetumor or cancer comprises a carcinoma, sarcoma, lymphoma, leukemia,adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma,mesothelioma, reticuloendothelial, lymphatic or haematopoieticneoplasia, tumor, cancer or malignancy.
 40. The method of claim 34 or35, wherein the sarcoma comprises a lymphosarcoma, liposarcoma,osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma orfibrosarcoma.
 41. The method of claim 39, wherein the haematopoieticneoplasia, tumor, cancer or malignancy comprises a myeloma, lymphoma orleukemia.
 42. The method of claim 34 or 35, wherein the tumor or cancercomprises a lung, thyroid, head or neck, nasopharynx, throat, nose orsinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid,lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum,jejunum (small intestine), colon, rectum), genito-urinary tract (uterus,ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney,pancreas, liver, bone, bone marrow, lymph, blood, muscle, or skinneoplasia, tumor, or cancer.
 43. The method of claim 34 or 35, whereinthe tumor or cancer comprises a breast cancer, prostate cancer, pancreascancer, gastric cancer, pleural mesothelioma, colon cancer, rectalcancer, large bowel cancer, small intestinal cancer, esophageal cancer,duodenal cancer, lingual cancer, pharyngeal cancer, salivary glandcancer, cerebral tumor, schwanoma, liver cancer, kidney cancer, bileduct cancer, endometrial cancer, cervical cancer, uterine body cancer,ovarian cancer, bladder cancer, urethral cancer, skin cancer, angioma,malignant lymphoma, malignant melanoma, thyroid cancer, parathyroidcancer, nasal cancer, paranasal cancer, auditory organ cancer, carcinomaof oral floor, laryngeal cancer, parotid cancer, submandibular cancer,bone tumor, angiofibroma, retinal sarcoma, penile cancer, testiculartumor, pediatric solid cancer, Kaposi's sarcoma, tumor of maxillarysinus, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, lymphoma,multiple myeloma or leukemia.
 44. The method of claim 1, wherein themammal is a human.
 45. The method of claim 1, wherein the peptidecompound comprises:(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg), or(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha).
 46. Themethod of claim 1, wherein the amount of the peptide compoundadministered is effective to treat the tumor or cancer.
 47. The methodof claim 1, wherein the method inhibits or reduces relapse, growth,progression, worsening or metastasis of the tumor or cancer.
 48. Themethod of claim 1, wherein the method results in partial or completedestruction of the neoplastic, tumor, cancer or malignant cell mass,volume, size or numbers of cells, stimulating, inducing or increasingneoplastic, tumor, cancer or malignant cell necrosis, lysis orapoptosis, reducing neoplasia, tumor, cancer or malignancy volume size,cell mass, inhibiting or preventing progression or an increase inneoplasia, tumor, cancer or malignancy volume, mass, size or cellnumbers, or prolonging lifespan.
 49. The method of claim 1, wherein themethod results in reducing or decreasing severity, duration or frequencyof an adverse symptom or complication associated with or caused by theneoplasia, tumor, cancer or malignancy.
 50. The method of claim 1,wherein the method results in reducing or decreasing pain, discomfort,nausea, weakness or lethargy, or results in increased energy, appetite,improved mobility or psychological well being.